Method for producing preparation containing bioactive substance

ABSTRACT

A method for producing a preparation containing a bioactive substance, characterized in that it comprises forming a solid material containing the bioactive substance and a polymer, and contacting the solid material with a high pressure gas. The method allows the production of a preparation which is suppressed in excessive initial release of the bioactive substance immediately after the administration thereof, is capable of releasing a predetermined amount of the bioactive substance over a long period of time, and is extremely reduced in the deterioration of the bioactive substance and in the amount of a residual organic solvent.

This application is a 371 of PCT/JP01/10416, filed Nov. 29, 2001, whichclaims priority to Japanese application 2000-367183, filed Dec. 1, 2000.

TECHNICAL FIELD

The present invention relates to a method for producing a preparationcontaining a bioactive substance. More specifically, the presentinvention relates to a method for producing a preparation comprising abioactive substance, which is unstable to heat or solvents, and apolymer.

BACKGROUND ART

Peptidic or non-peptidic bioactive substances are known to exhibitvarious pharmacological activities in a living body, and the applicationthereof as medicaments have been attempted. However, it is required toadminister these bioactive substances frequently since their half-lifein a living body is generally short. Then, physical burden of patientsdue to administration by injection cannot be ignored. For example,growth hormone, a representative hormone which is originally producedand secreted in the anterior portion of the pituitary gland, is abioactive peptide having widely diverse physiological activities suchas, in addition to promotion of growth in the body, metabolism ofsaccharides and lipids, anabolism of proteins, cell proliferation anddifferentiation, and the like. At present, growth hormone is produced ona large scale by Escherichia coli using genetic recombinationtechnology, and put to medicinal use clinically and worldwide. However,it is required to administer growth hormone frequently in order tomaintain an effective blood level because of its short biologicalhalf-life. Especially, in the case of pituitary dwarfism, a dailysubcutaneous administration to infants or young patients over a longperiod of time ranging from a few months to 10 years or more is actuallytaken place.

In order to deal with problems inherent in such bioactive substances,various drug delivery systems have been studied. For example, asustained-release agent that provides sustained-release of a bioactivepeptide over a long period of time has been studied. JP 8-217691 A(WO96/07399) discloses a method for producing a sustained-releasepreparation comprising a water-insoluble or poorly water solublepolyvalent metal salt of a water-soluble peptidic bioactive substance,which is formed by an aqueous solution of zinc chloride, etc., and abiodegradable polymer.

Further, for a sustained-release preparation using a biodegradablepolymer, it is desired to maintain the activity of a bioactive substancewith suppressing the initial release of a bioactive substance, inparticular, release of the excess amount within one day, and to controlthe release of the bioactive substance arbitrarily over a long period oftime. Regarding this problem, JP 11-322631 A discloses a method forproducing a sustained-release preparation comprising adding awater-miscible organic solvent and/or a volatile salt to an aqueoussolution of a bioactive peptide, followed by lyophilizing to obtain abioactive peptide powder, dispersing the powder in a solution of abiodegradable polymer in an organic solvent, and removing the organicsolvent. Furthermore, JP 9-132524 A discloses a method for producingsustained-release microcapsules comprising a bioactive substance and abiodegradable polymer which comprises, after forming microcapsules,heat-drying the microcapsules at a temperature of not less than theglass transition temperature of the biodegradable polymer for about 24to 120 hours. These are methods for producing a sustained-releasepreparation containing very little residual organic solvent and havingvery superior clinical properties as medicaments.

OBJECTS OF THE INVENTION

However, according to the solvent-removing procedures in theabove-mentioned production methods, since it takes long period of timefor removing the solvent, there is still room for improvement in view ofthe production costs for the industrial application.

On the other hand, as a procedure for removing a solvent that remains ina component (e.g., polymer) used for formulating a preparation of amedicament, heat drying method, vacuum drying method and flash dryingwith dried gas have been known. However, in these procedures, when asubstance has strong affinity for a solvent and is unstable to heat, theremoval of the solvent tends to be insufficient or, in some cases, thesubstance is decomposed. Furthermore, in these procedures, when theboiling point of a solvent to be removed is high, the properties of apreparation obtained may be deteriorated.

SUMMARY OF THE INVENTION

The present inventors have studied intensively to solve theabove-mentioned problems and unexpectedly found that, in a method forproducing a sustained-release preparation comprising a bioactivesubstance and a biodegradable polymer, a sustained-release preparationhaving superior clinical properties as a medicament, in which excessinitial release of the bioactive substance immediately afteradministration is markedly suppressed, a constant amount of thebioactive substance is being released from immediately afteradministration over a long period of time and very little residualorganic solvent is contained therein, can be obtained by, after forminga solid material, contacting the solid material with high-pressure gasfor about 10 minutes to about 12 hours. The present invention has beencompleted based on these findings.

That is, the present invention provides:

(1) A method for producing a preparation containing a bioactivesubstance, which comprises forming a solid material containing thebioactive substance and a polymer, and contacting the solid materialwith high-pressure gas;

(2) The method according to the above (1), wherein the bioactivesubstance is that being unstable to heat or solvents;

(3) The method according to the above (1), wherein the bioactivesubstance is a bioactive peptide having a molecular weight of about2,000 to about 500,000;

(4) The method according to the above (1), wherein the bioactivesubstance is a bioactive peptide having a molecular weight of about5,000 to about 500,000;

(5) The method according to the above (4), wherein the bioactivesubstance is human growth hormone;

(6) The method according to the above (1), wherein the bioactivesubstance is a non-peptidic compound;

(7) The method according to the above (6), wherein the non-peptidiccompound is a compound having an oxygen atom in the molecule;

(8) The method according to the above (6), wherein the non-peptidiccompound is a compound having an ether bond or a carbonyl group;

(9) The method according to the above (6), wherein the non-peptidecompound is a compound represented by the formula (I):

wherein R¹ represents a group capable of forming an anion or a groupwhich may be converted into said group, X represents that the phenylenegroup and the phenyl group are linked directly or via a spacer of anatomic chain having two or less atom(s), n represents an integer of 1 or2, ring A represents a benzene ring which may be further substituted, R²represents a group capable of forming an anion or a group which may beconverted into said group, R³ represents a hydrocarbon residue which maylink via a heteroatom and may be substituted, or a salt thereof;

(10) The method according to the above (6), wherein the non-peptidiccompound is losartan, eprosartan, candesartan cilexetil, candesartan,valsartan, telmisartan, irbesartan, tasosartan or olmesartan;

(11) The method according to the above (6), wherein the non-peptidecompound is candesartan;

(12) The method according to the above (1), wherein the polymer isbiodegradable;

(13) The method according to the above (12), wherein the biodegradablepolymer is a homopolymer or a copolymer of α-hydroxycarboxylic acids, ora mixture thereof;

(14) The method according to the above (13), wherein the biodegradablepolymer is a homopolymer or a copolymer of lactic acid/glycolic acidhaving a composition ratio of lactic acid/glycolic acid of about 100/0to about 40/60 mol %;

(15) The method according to the above (13), wherein the biodegradablepolymer is a homopolymer of lactic acid;

(16) The method according to the above (12), wherein the weight-averagemolecular weight of the biodegradable polymer is about 3,000 to about50,000;

(17) The method according to the above (1), wherein the solid materialis contacted with high-pressure gas at a temperature range of about +20°C. to about −60° C. based on the glass transition temperature of thepolymer;

(18) The method according to the above (17), wherein the solid materialis contacted with high-pressure gas at a temperature range of about +0°C. to about −40° C. based on the glass transition temperature of thepolymer;

(19) The method according to the above (1), wherein the period forcontacting the solid material with high-pressure gas is about 5 minutesto about 48 hours;

(20) The method according to the above (19), wherein the period forcontacting the solid material with high-pressure gas is about 10 minutesto about 12 hours;

(21) The method according to the above (1), wherein the high-pressuregas is inert to the bioactive substance and polymer;

(22) The method according to the above (21), wherein the high-pressuregas is carbon dioxide;

(23) The method according to the above (1), wherein the pressure of thehigh-pressure gas is about 1 MPa to about 7 MPa;

(24) The method according to the above (23), wherein the pressure of thehigh-pressure gas is about 1 MPa to about 4 MPa;

(25) The method according to the above (23), wherein the preparation issustained-release microcapsules;

(26) The method according to the above (25), wherein thesustained-release microcapsules are obtained by drying-in-water method;

(27) A preparation obtained by the method according to the above (1);

(28) Sustained-release microcapsules obtained by the method according tothe above (25);

(29) An injectable preparation comprising the sustained-releasemicrocapsules according to the above (28);

(30) A method for suppressing the initial release of a bioactivesubstance, which comprises forming a solid material containing saidbioactive substance and a polymer, and contacting the solid materialwith high-pressure gas; and

(31) A method for suppressing the denaturation of a bioactive substance,which comprises forming a solid material containing said bioactivesubstance and a polymer, and contacting the solid material withhigh-pressure gas.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of a solvent-removing apparatus usingcarbon dioxide in a high-pressure gaseous state.

Each number in the drawing has the following meaning.

1: liquefied carbon dioxide bomb, 2: CO₂ delivery pump, 3: heatexchanger, 4: extraction vessel, 5: thermostat, 6: detector, 7:automatic pressure regulating valve, 8: recovery vessel.

DETAILED DESCRIPTION OF THE INVENTION

The bioactive substance used in the present invention includes variousdrugs, which have useful physiological activities for animals and plantsand can be used as an agrochemical or an animal drug, or can be usedclinically. As the bioactive substance used in the present invention, abioactive substance being unstable to heat or solvents is preferred. Thebioactive substance being unstable to heat or solvents used herein meansa bioactive substance that is decomposed, metabolized, inactivated ordenatured in a production step which involves heating or contact with anorganic solvent, such as emulsification, removing of a solvent ordrying. The agrochemical includes, for example, control agents forpests, control agents for plant diseases, herbicides, plant growthregulators, fertilizers and the like, and the animal drug includes, forexample, antibacterial agents, vitamin preparations, hormonepreparations, vaccines, additives for fishery products, insecticide anddisinfectant preparations, drugs for pets and the like. For an idealagrochemical or animal drug, which is safe and environment-friendly,reduction of residual solvents is important. Examples of various drugsthose can be used clinically include, and are not specifically limited,peptidic compounds having physiological activities, as well asantibiotics, antifungal agents, antihyperlipidemic agents, antitumoragents, antipyretic agents, analgesic agents, antiinflammatory agents,antitussive and expectorant agents, sedatives, muscle relaxants,antiepileptics, antiulcer agents, antidepressants, antiallergic agents,cardiotonics, antiarrhythmic agents, vasodilators, hypotensivediuretics, antidiabetic agents, anticoagulants, hemostatic agents,antiplatelet agents, antituberculous agent, hormones, antinarcotics,bone resorption-suppressing agents, osteogenesis-accelerating agents,and neovascularization suppressing agents. Of these, a peptidic ornon-peptidic bioactive substance that produces a dimer, a polymer orrelated substances such as an oxidized substances, deamidatedsubstances, and the like by heat, or a peptidic or non-peptidicbioactive substance that produces a reaction product with abiodegradable polymer, is preferably used in the present invention.

The bioactive peptide in the present invention includes various peptidesor proteins, which have physiological activities useful for mammals andcan be used clinically. As the “bioactive peptide”, that having amolecular weight (as monomers) of, for example, about 200 to 500,000,preferably molecular weight of about 2,000 to 500,000, is generallyused. More preferably, a peptide having a molecular weight of 5,000 toabout 500,000 is used.

The typical activity of the bioactive peptide includes hormoneactivities. The bioactive peptide may be any of natural substances,synthesized substances and semi-synthesized substances, or may bederivatives or related substances thereof. The functional mechanism ofthe bioactive peptide may be either of agonistic and antagonistic.

As the bioactive peptide in the present invention, there can be usedpeptide hormones, cytokines, peptide nerve transmitter substances,hematopoietic factors, various growth factors, enzymes, polypeptideantibiotics, analgetic peptides, vaccines, and the like.

As the peptide hormones, there can be used insulin, somatostatin,somatostatin derivatives (Sandostatin; see U.S. Pat. Nos. 4,087,390,4,093,574, 4,100,117 and 4,253,998), growth hormones (GH), sodiumdiuretic peptides, gastrin, prolactin, adrenocorticotropic hormone(ACTH), ACTH derivatives (e.g., ebiratide, etc.), melanocyte-stimulatinghormone (MSH), thyroid hormone-releasing hormone (TRH) and salts andderivatives thereof (see JP 50-121273 A and JP 52-116465 A),thyroid-stimulating hormone (TSH), luteinizing hormone (LH),follicle-stimulating hormone (FSH), human chorionic gonadotropin (HCG),thymosin, motilin, vasopressin, vasopressin derivatives [desmopressin,see Folia Endocrinologica Japonica, Vol. 54, No. 5, pp. 676–691 (1978)],oxytocin, calcitonin, parathyroid hormone (PTH), glucagon, secretin,pancreozymin, cholecystokinin, angiotensin, human placental lactogen,glucagon-like peptide (GLP-1) and derivatives thereof (see JP 6-80584 A,JP 7-2695 A, EP 658568, JP 8-245696 A, JP 8-269097 A, WO97/15296,WO97/31943, WO98/19698, WO98/43658, JP 10-511365 A, WO99/55310, JP11-513983 A, CA2270320, WO99/64061, JP 11-514972 A, JP 2000-500505 A,WO2000/66138, WO2000/66142, WO2000/78333, JP 2001-11095, Tissue Eng.7(1)35–44(2001), Diabetologia 43(10)1319–1328(2000), WO2000/34331,WO2000/34332, U.S. Pat. No. 6,268,343, U.S. 2001011071 A, U.S.2001006943 A, EP 0733644, WO2000/77039, WO99/43707, WO99/43341,WO99/43706, WO99/43708, WO99/43705, WO99/29336, WO2000/37098, EP0969016, U.S. Pat. No. 5,981,488, U.S. Pat. No. 5,958,909, WO93/25579,WO98/43658, EP 0869135, U.S. Pat. Nos. 5,614,492, 5,545,618, 5,120,712,5,118,666, WO95/05848, WO91/11457, EP 0708179, WO96/06628, EP0658568,WO87/06941), glucose-dependent insulin secretory peptide (GIP), exendinand derivatives thereof (see WO2000/66629, WO2000/41546, WO99/07404,WO2000/09666, and U.S. Pat. No. 5,424,286), metastin and derivativesthereof (see WO2000/24890), and the like. Preferably, the peptidehormone is insulin and growth hormone, etc.

Growth hormone (hereinafter referred to as GH) originating from anyanimal species can be used, and is preferably human growth hormone(hereinafter referred to as hGH). Further, although natural productsextracted from the pituitary gland can be used in the present invention,genetic recombinant type GH (see JP 6-12996 B and JP 6-48987 B) ispreferred. The recombinant type hGH having the same structure as anatural type which does not have methionine at the N-terminal group ismore preferred. Such GH may be in the form of a metal salt, and onebeing substantially free from a metal is also used. About 20K daltontype of hGH (see JP 7-101877 A and JP 10-265404 A) as well as about 22Kdalton type of hGH can be used. Furthermore, the derivatives or relatedsubstances of hGH (see WO99/03887) can be used.

As the cytokines, for example, lymphokines, monokines, and the like canbe used. As the lymphokines, there can be used, for example, interferons(alpha type, beta type, gamma type and the like), interleukins (IL-2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 and the like) and the like. As themonokines include, for example, interleukin-1 (IL-1), tumor necrosisfactor (TNF), and the like can be used. The cytokines are preferablylymphokines, and the like, more preferably interferons, and the like,especially preferably interferon-alpha.

As the peptide neurotransmitters, substance P, serotonin, GABA, and thelike can be used.

As the hematopoietic factors, there can be used, for example,erythropoietin (EPO), colony stimulating factors (G-CSF, GM-CSF, M-CSFand the like), thrombopoietin (TPO), platelet growth stimulating factor,megakaryocyte potentiator, and the like.

As various growth factors, there can be used, for example, basic andacidic fibroblast growth factors (FGF) and their family (e.g., EGF,TGF-α, TGF-β, PDGF, acidic FGF, basic FGF, FGF-9, and the like), nervegrowth factors (NGF) and their family (e.g., BDNF, NT-3, NT-4, CNTF,GDNF, and the like), insulin-like growth factors (e.g. IGF-1, IGF-2, andthe like), bone morphogenetic protein (BMP) and their family, and thelike.

As the enzymes include, there can be used, for example, superoxidedismutase (SOD), urokinase, tissue plasminogen activator (TPA),asparaginase, kallikrein, and the like.

As the polypeptide antibiotics, for example, polymixin B, colistin,gramicidin, bacitracin, and the like can be used.

As the analgesic peptides, for example, enkephalin, enkephalinderivatives (see U.S. Pat. No. 4,277,394 and EP 031567 A), endorphin,kyotorphin, and the like can be used.

As the vaccines, there can be used, for example, influenza vaccine,Japanese encephalitis vaccine, antirabies vaccine, hepatitis B vaccine,hepatitis A vaccine, cholera vaccine, DPT mixed vaccine, pneumococcusvaccine, diphteria vaccine, tetanus vaccine, polio vaccine, prostaticspecific antigen vaccine, and the like.

Furthermore, the bioactive peptides include thymopoietin, dynorphin,bombesin, caerulein, thymostimulin, thymic humoral factor (THF), bloodthymic factor (FTS) and derivatives thereof (see U.S. Pat. No.4,229,438), other thymic factors [Igaku no Ayumi, Vol.125, No. 10, pp.835–843 (1983)], neurotensin, bradykinin, endothelin-antagonisticpeptides (see EP 436189 A, EP 457195 A and EP 496452 A, JP 3-94692 A andJP 3-130299 A), and the like.

In the present invention, when the bioactive peptide contains a metal,the metal contained in the bioactive peptide may be removed previously,if necessary, and, as a method for removing the metal, a known methodcan be used. For example, an insulin in the form of amorphous andcontaining a minimum amount of metal can be obtained by dialyzing anaqueous solution of insulin acidified with hydrochloric acid to water ora solution of ammonium acetate, followed by lyophilization.

As the non-peptidic bioactive substance of the present invention, forexample, there may be mentioned one or more components selected fromnourishing and health-promoting agents,antipyretic-analgesic-antiinflammatory agents, antipsychotic drugs,antianxiety drugs, antidepressants, hypnotic-sedatives, spasmolytics,central nervous system affecting drugs, cerebral metabolismameliolators, antiepileptics, sympathomimetic agents, gastrointestinalfunction conditioning agents, antacids, antiulcer agents,antitussive-expectorants, antiemetics, respiratory stimulants,bronchodilators, antiallergic agents, dental buccal drugs,antihistamines, cardiotonics, antiarrhythmic agents, diuretics,hypotensive agents, vasoconstrictors, coronary vasodilators, peripheralvasodilators, antihyperlipidemic agents, cholagogues, antibiotics,chemotherapeutic agents, antidiabetic agents, drugs for osteoporosis,skeletal muscle relaxants, antimotion sickness drugs, hormones, alkaloidnarcotics, sulfa drugs, drugs for treatment of gout, anticoagulants,anti-malignant tumor agents, agents for Alzheimer's disease and thelike.

Examples of the nourishing and health-promoting agents include vitaminssuch as vitamin A, vitamin D, vitamin E (d-α-tocopherol acetate and thelike), vitamin B₁ (dibenzoylthiamine, fursultiamine hydrochloride andthe like), vitamin B₂ (riboflavin butyrate and the like), vitamin B₆(pyridoxine hydrochloride and the like), vitamin C (ascorbic acid,sodium L-ascorbate and the like), vitamin B₁₂ (hydroxocobalamin acetateand the like) and the like; minerals such as calcium, magnesium andiron; amino acids; oligosaccharides; galenical; and the like. Examplesof the antipyretic-analgesic-antiinflammatory agents include aspirin,acetaminophen, ethenzamide, ibuprofen, diphenhydramine hydrochloride,dl-chlorpheniramine maleate, dihydrocodeine phosphate, noscapine,methylephedrine hydrochloride, phenylpropanolamine hydrochloride,caffeine, anhydrous caffeine, tolfenamic acid, mefenamic acid,diclofenac sodium, flufenamic acid, salicylamide, aminopyrine,ketoprofen, indomethacin, bucolome, pentazocine and the like. Examplesof the antipsychotic drugs include chlorpromazine, reserpine and thelike. Examples of the antianxiety drugs include alprazolam,chlordiazepoxide, diazepam and the like. Examples of the antidepressantsinclude imipramine, maprotiline, amphetamine and the like.

Examples of the hypnotic-sedatives include estazolam, nitrazepam,diazepam, perlapine, phenobarbital sodium and the like. Examples of thespasmolytics include scopolamine hydrobromide, diphenhydraminehydrochloride, papaverine hydrochloride and the like. Examples of thecentral nervous system affecting drugs include citicoline, rotirenineand the like. Examples of the cerebral metabolism ameliolators includevinpocetine, meclofenoxate hydrochloride and the like. Examples of theantiepileptics include phenytoin, carbamazepine and the like. Examplesof the sympathomimetic agents include isoproterenol hydrochloride andthe like. Examples of the gastrointestinal function conditioning agentsinclude stomachic-digestives such as gentian, swertia herb, nux vomica,phellodendron bark, bitter orange peel, Condurango, cinnamon oil and thelike; intestinal function controlling drugs such as perperinehydrochloride, resistant lactic acid bacterium, Lactobacillus bifidusand the like. Examples of the antacids include magnesium carbonate,sodium hydrogen carbonate, magnesium aluminometasilicate, synthetichydrotalcite, precipitated calcium carbonate, magnesium oxide and thelike. Examples of the antiulcer agents include lansoprazole, omeprazole,rabeprazole, pantoprazole, famotidine, cimetidine, ranitidinehydrochloride and the like.

Examples of the antitussive-expectorants include chloperastinehydrochloride, dextromethorphan hydrobromide, theophylline, potassiumguaiacolsulfonate, guaifenesin, codeine phosphate and the like. Examplesof the antiemetics include diphenidol hydrochloride, metoclopramide andthe like. Examples of the respiratory stimulants include levallorphantatrate and the like. Examples of the bronchodilators includetheophylline, salbutamol sulfate and the like. Examples of theantiallergic agents include amlexanox, seratrodast and the like.Examples of the dental buccal drugs include oxytetracycline,triamcinolone acetonide, chlorhexidine hydrochloride, lidocaine and thelike. Examples of the antihistamines include diphenhydraminehydrochloride, promethazine, isothipendyl hydrochloride,dl-chlorpheniramine maleate and the like. Examples of the cardiotonicsinclude caffeine, digoxin and the like. Examples of the antiarryhythmicagents include procainamide hydrochloride, propranolol hydrochloride,pindolol and the like. Examples of the diuretics include isosorbide,furosemide and the like. Examples of the hypotensive agents includedelapril hydrochloride, captopril, hexamethonium bromide, hydralazinehydrochloride, labetalol hydrochloride, manidipine hydrochloride,losartan, eprosartan, candesartan cilexetil (TCV-116), candesartan(CV-11974), valsartan, telmisartan, irbesartan, tasosartan, ormesartanand the like. As the hypotensive agents, candesartan, candesartancilexetil and the like are preferred, and candesartan and the like arespecifically preferred.

Examples of the vasoconstrictors include phenylephrine hydrochloride andthe like. Examples of the coronary vasodilators include carbocromenhydrochloride, molsidomine, verapamil hydrochloride and the like.Examples of the peripheral vasodilators include cinnarizine and thelike. Examples of the antihyperlipidemic agents include cerivastatinsodium, simvastatin, pravastatin sodium and the like. Examples of thecholagogues include dehydrocholic acid, trepibutone and the like.Examples of the antibiotics include cephem antibiotics such ascefalexin, amoxicillin, pivmecillinam hydrochloride, cefotiamdihydrochloride, cefozopran hydrochloride, cefinenoxime hydrochloride,cefsluodin sodium, etc.; synthetic antibacterials such as ampicillin,cyclacillin, sulbenicillin sodium, nalidixic acid, enoxacin, etc.;monobactam antibiotics such as carumonam sodium; penem antibiotics,carbapenem antibiotics, etc.; and the like. Examples of thechemotherapeutic agents include sulfamethizole hydrochloride,thiazosulfone and the like. Examples of the antidiabetic agents includetolbutamide, voglibose, pioglitazone (hydrochloride), troglitazone,5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]methyl]-2,4-thiazolidinedione(BRL-49653), acarbose, miglitol, emiglitate and the like. Examples ofthe drugs for osteoporosis include ipriflavone and the like. Examples ofthe skeletal muscle relaxants include methocarbamol and the like.Examples of the antimotion sickness drugs include meclizinehydrochloride, dimenhydrinate and the like.

Examples of the hormones include riothyroinine sodium, dexamethasonesodium phosphate, prednisolone, oxendolone, leupororelin acetate and thelike. Examples of the alkaloid narcotics include opium, morphinehydrochloride, ipecac, oxycodone hydrochloride, opium alkaloidshydrochlorides, cocaine hydrochloride and the like. Examples of thesulfa drugs include sulfanilamide, sufamethizole and the like. Examplesof the drugs for treatment of gout include allopurinol, colchicine andthe like. Examples of the anticoagulants include dicoumarol and thelike. Examples of the anti-malignant tumor agents include5-fluorouracil, uracil, mitomycin and the like. Examples of the agentsfor Alzheimer's disease include idebenone, vinpocetine and the like.

As the non-peptidic bioactive substance in the present invention, acompound having an oxygen atom in the molecule, specifically, a compoundhaving an ether bond or a carbonyl group is preferred. Such compoundincludes a compound represented by the formula (I):

or a salt thereof.

In the above-mentioned formula (I), examples of the group capable offorming an anion (a group having hydrogen atom that may be liberated asa proton) as R¹ include (1) a carboxyl group, (2) a tetrazolyl group,(3) a trifluoromethanesulfonic acid amide group (—NHSO₂CF₃), (4) aphosphoric acid group, (5) a sulfonic acid group, (6) an optionallysubstituted 5- to 7-membered (preferably 5- or 6-membered) monocyclicheterocycle residue containing one or two or more of N, S and O, and thelike.

Examples of the above-mentioned “optionally substituted 5- to 7-membered(preferably 5- or 6-membered) monocyclic heterocycle residue containingone or two or more of N, S and O” include

and the like. Furthermore, when the g in the above-mentioned formularepresents —NH— and the like, the linkage between the heterocyclicresidue represented by R¹ and the phenyl group to which the heterocyclicresidue is linked, may link via one of existing plural nitrogen atoms,in addition to the above-mentioned carbon-carbon bond(s). For example,when R¹ is represented by the formula:

specifically, examples thereof are

and the like, respectively. The other examples of the link via anitrogen atom include

and the like.

In the above-mentioned formula, g represents —CH₂—, —NH—, —O— or—S(O)_(m)—, >=Z, >=Z′ and >=Z″ each represents a carbonyl group, athiocarbonyl group or an optionally oxidized sulfur atom (e.g., S, S(O),S(O)₂ and the like) (preferably a carbonyl or a thiocarbonyl group, morepreferably a carbonyl group), respectively, and m represents 0, 1 or 2.

The heterocyclic residue represented by R¹ is preferably groups obtainedby eliminating one hydrogen atom from a ring that has both —NH— or —OHgroup as a proton donor, and a carbonyl group, a thiocarbonyl group, asulfinyl group, or the like as a proton acceptor, simultaneously, suchas an oxadiazolone ring, an oxadiathiazolone ring, a thiadiazolonegroup, or the like. Furthermore, the heterocyclic residue represented byR¹ may form a condensed ring group by linking substituents on the ring.As the heterocyclic residue represented by R¹, a 5- or 6-membered ringresidue is preferred, and a 5-membered residue is more preferred.

As the heterocyclic residue represented by R¹, a group represented bythe formula:

wherein i represents —O— or —S—, j represents >=O, >=S or >=S(O)_(m), mis as defined above (among these,2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl,2,5-dihydro-5-thioxo-1,2,4-oxadiazol-3-yl,2,5-dihydro-5-oxo-1,2,4-thiadiazol-3-yl, especially2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl) and the like are preferred.

Furthermore, the above-mentioned heterocyclic residue (R¹) has tautomersas shown below. For example, when Z=O and g=O in the following formula:

three tautomers a′, b′ and c′, such as

are present, and the heterocyclic residue represented by the formula:

encompasses all of the above-mentioned a′, b′ and c′.

The group capable of forming an anion as R¹ may be protected with anoptionally substituted lower (C₁₋₄) alkyl group or an acyl group (e.g.,a lower (C₂₋₅) alkanoyl, a benzoyl, etc.), and the like, atsubstitutable position(s).

The optionally substituted lower (C₁₋₄) alkyl group include, forexample, (1) a lower (C₁₋₄) alkyl group optionally substituted with 1 to3 phenyl group(s) optionally having a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like (e.g., methyl, triphenylmethyl,p-methoxybenzyl, p-nitrobenzyl and the like), (2) a lower (C₁₋₄)alkoxy-lower (C₁₋₄) alkyl group (e.g., methoxymethyl, ethoxymethyl andthe like), (3) the formula —CH(R⁴)—OCOR⁵ [wherein R⁴ represents (a)hydrogen, (b) a straight chain or branched lower alkyl group having 1–6carbon atom(s) (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl and the like), (c) astraight chain or branched lower alkenyl group having 2–6 carbon atomsor (d) a cycloalkyl group having 3–8 carbon atoms (e.g., cyclopentyl,cyclohexyl, cycloheptyl and the like), R⁵ represents (a) a straightchain or branched lower alkyl group having 1–6 carbon atom(s) (e.g.,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,t-butyl, n-pentyl, isopentyl, neopentyl and the like), (b) a straightchain or branched lower alkenyl group having 2–6 carbon atoms, (c) alower alkyl group having 1 to 3 carbon atom(s) substituted with acycloalkyl group having 3–8 carbon atoms (e.g., cyclopentyl, cyclohexyl,cycloheptyl and the like) or an optionally substituted aryl group (e.g.,a phenyl or naphthyl group and the like, each of which may have ahalogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and thelike) (e.g., benzyl, p-chlorobenzyl, phenethyl, cyclopentylmethyl,cyclohexylmethyl and the like), (d) a lower alkenyl group having 2 to 3carbon atoms substituted by a cycloalkyl having 3–8 carbon atoms or anoptionally substituted aryl group (e.g., a phenyl or naphthyl group andthe like, each of which may have a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group having alkenylportion(s) such as vinyl, propenyl, allyl, isopropenyl and the like,such as cinnamyl, and the like), (e) an optionally substituted arylgroup (e.g., a phenyl or a naphthyl group and the like, each of whichmay have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxyand the like, such as phenyl, p-tolyl, naphthyl and the like), (f) astraight or a branched lower alkoxy group having 1–6 carbon atom(s)(e.g., methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, t-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy and thelike), (g) a straight chain or branched lower alkenyloxy group having 2to 8 carbon atoms (e.g., allyloxy, isobutenyloxy and the like), (h) acycloalkyloxy group having 3–8 carbon atoms (e.g., cyclopentyloxy,cyclohexyloxy, cycloheptyloxy and the like), (i) a lower alkoxy grouphaving 1 to 3 carbon atom(s) substituted with a cycloalkyl having 3–8carbon atoms (e.g., cyclopentyl, cyclohexyl, cycloheptyl and the like)or an optionally substituted aryl group (e.g., a phenyl or naphthylgroup and the like, each of which may have a halogen atom, nitro, lower(C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group havingalkoxy portion(s) such as methoxy, ethoxy, n-propoxy, isopropoxy and thelike, such as benzyloxy, phenethyloxy, cyclopentylmethoxy,cyclohexylmethoxy and the like), (j) a lower alkenyloxy group having 2to 3 carbon atoms substituted with a cycloalkyl having 3–8 carbon atoms(e.g., cyclopentyl, cyclohexyl, cycloheptyl and the like) or anoptionally substituted aryl group (e.g., a phenyl or naphthyl group andthe like, each of which may have a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group havingalkenyloxy portion(s) such as vinyloxy, propenyloxy, allyloxy,isopropenyloxy and the like, such as cinnamyloxy and the like, and thelike), (k) an optionally substituted aryloxy group (e.g., phenoxy ornaphthoxy group and the like, each of which may have a halogen atom,nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and the like, such asphenoxy, p-nitrophenoxy, naphthoxy and the like, and the like)], and thelike.

Furthermore, the group capable of forming an anion as R¹ may havesubstituent(s) such as an optionally substituted lower (C₁₋₄) alkylgroup (which includes the groups similar to the “optionally substitutedlower (C₁₋₄) alkyl group” that is exemplified as protective groups forthe group capable of forming an anion as the above-mentioned R¹), ahalogen atom, nitro, cyano, lower (C₁₋₄) alkoxy, an amino optionallysubstituted with 1 or 2 of lower (C₁₋₄) alkyl(s) and the like, at thesubstitutable position(s), in addition to the protective groups such asthe above-mentioned optionally substituted lower (C₁₋₄) alkyl group oracyl group (e.g., a lower (C₂₋₅) alkanoyl, a benzoyl and the like).

In the above-mentioned formula, the group which may be converted intothe group capable of forming an anion as R¹ (a group having hydrogenatom that may be liberated as a proton) may be a group which can beconverted into the group capable of forming an anion by a reaction underbiological, i.e., physiological conditions (for example, an in vivoreaction such as oxidation, reduction, hydrolysis or the like with an invivo enzyme, and the like) (so-called prodrug), or may be a group whichcan be converted into a group capable of forming an anion represented byR¹ by a chemical reaction, such as cyano, an N-hydroxycarbamimidoylgroup (—C(═N—OH)—NH₂), or (1) a carboxyl group, (2) a tetrazolyl group,(3) a trifluoromethanesulfonic acid amide group (—NHSO₂CF₃), (4) aphosphoric acid group, (5) a sulfonic acid group, and (6) an optionallysubstituted 5- to 7-membered (preferably 5-or 6-membered) monocyclicheterocycle residue containing one or two or more of N, S and O, each ofwhich has been protected with an optionally substituted lower (C₁₋₄)alkyl group or an acyl group (so-called synthetic intermediate).

Preferred examples of R¹ include a carboxyl, a tetrazolyl, or a2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl (preferably tetrazolyl)optionally protected with an optionally substituted lower (C₁₋₄) alkyl(e.g., methyl, triphenylmethyl, methoxymethyl, ethoxymethyl,p-methoxybenzyl, p-nitrobenzyl and the like) or an acyl group (e.g.,lower (C₂₋₅) alkanoyl, benzoyl and the like), or a cyano, anN-hydroxycarbamimidoyl (preferably cyano), and specifically, cyano ispreferably used.

In the above-mentioned formula, X represents that the adjacent phenylenegroup and phenyl group are linked directly or via a spacer of an atomicchain having two or less atom(s) (preferably linked directly), and thespacer of an atomic chain having two or less atom(s) may be any divalentchain whose straight chain portion is constituted of 1 or 2 atom(s). Thechain may also have side chain(s). Specifically, it includes a lower(C₁₋₄) alkylene whose straight chain portion is constituted of 1 or 2atoms, —CO—, —O—, —S—, —NH—, —CO—NH—, —O—CH₂—, —S—CH₂—, —CH═CH— and thelike.

In the above-mentioned formula, n represents an integer of 1 or 2(preferably 1).

In the above-mentioned formula, ring A represents a benzene ring whichmay be further substituted in addition to the substituent R², andexamples of the substituent include (1) halogen (e.g., F, Cl, Br and thelike), (2) cyano, (3) nitro, (4) optionally substituted lower (C₁₋₄)alkyl, (5) lower (C₁₋₄) alkoxy, (6) an optionally substituted aminogroup (e.g., amino, N-lower (C₁₋₄) alkylamino (e.g., methylamino and thelike), N,N-dilower (C₁₋₄) alkylamino (e.g., dimethylamino and the like),N-arylamino (e.g., phenylamino and the like), alicyclic amino (e.g.,morpholino, piperidino, piperadino, N-phenylpiperadino and the like),and the like), (7) a group represented by the formula —CO-D′ [wherein D′represents a hydroxy group or lower (C₁₋₄) alkoxy in which the alkylportion may be substituted with a hydroxy group, lower (C₁₋₄) alkoxy,lower (C₂₋₆) alkanoyloxy (e.g., acetoxy, pivaloyloxy and the like),lower (C₁₋₆) alkoxycarbonyloxy (e.g., methoxycarbonyloxy,ethoxycarbonyloxy and the like) or lower (C₃₋₆) cycloalkoxycarbonyloxy(e.g., cyclohexyloxycarbonyloxy and the like)], (8) tetrazolyl, atrifluoromethanesulfonic acid amide group, a phosphoric acid group or asulfonic acid group, each of which is optionally protected withoptionally substituted lower (C₁₋₄) alkyl (including the groups similarto the “optionally substituted lower (C₁₋₄) alkyl group” exemplified asprotective groups for the group capable of forming an anion as theabove-mentioned R¹) or acyl (e.g., lower (C₂₋₅) alkanoyl, benzoyl andthe like), or the like.

One or two of these substituents may be present at the substitutableposition(s) of the benzene ring, simultaneously, while preferredsubstituent which is further possessed by ring A in addition to thesubstituent R² is optionally substituted lower (C₁₋₄) alkyl (e.g., lower(C₁₋₄) alkyl optionally substituted with a hydroxy group, a carboxylgroup, halogen, etc., and the like), halogen, and the like, and morepreferably, the ring A does not have any substituent except for thesubstituent R².

In the above-mentioned formula, examples of the group capable of formingan anion as R² (a group having hydrogen atom that may be liberated as aproton) include (1) a carboxyl group which may be esterified oramidated, (2) a tetrazolyl group, (3) a trifluoromethanesulfonic acidamide group (—NHSO₂CF₃), (4) a phosphoric acid group, (5) a sulfonicacid group, and the like. These groups may be protected with anoptionally substituted lower alkyl group (including a group similar tothe “optionally substituted lower (C₁₋₄) alkyl group” exemplified asprotective groups for the group capable of forming an anion as theabove-mentioned R¹) or an acyl group (e.g., lower (C₂₋₅) alkanoyl,benzoyl, and the like), and may be any group as long as it is a groupcapable of forming an anion or a group which may be converted into sucha group under biological, i.e., physiological conditions (for example,an in vivo reaction such as oxidation, reduction or hydrolysis, and thelike with an in vivo enzyme, and the like), or chemically.

Examples of the carboxyl which may be esterified or amidated, as R²,include a group represented by the formula —CO-D [wherein D represents(1) a hydroxy group, (2) optionally substituted amino (for example,amino, N-lower (C₁₋₄) alkylamino, N,N-dilower (C₁₋₄) alkylamino and thelike), (3) optionally substituted alkoxy {e.g., (i) a lower (C₁₋₆)alkoxy group in which the alkyl portion is optionally substituted with ahydroxy group, optionally substituted amino (e.g., amino, N-lower (C₁₋₄)alkylamino, N,N-dilower (C₁₋₄) alkylamino, piperidino, morpholino andthe like), halogen, lower (C₁₋₆) alkoxy, lower (C₁₋₆) alkylthio, lower(C₃₋₈) cycloalkoxy or optionally substituted dioxolenyl (e.g.,5-methyl-2-oxo-1,3-dioxolen-4-yl and the like), or (ii) a group of theformula —O—CH(R⁶)—OCOR⁷ [wherein R⁶ represents (a) hydrogen, (b) astraight chain or branched lower alkyl group having 1–6 carbon atom(s)(e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,n-pentyl, isopentyl, neopentyl and the like), (c) a straight chain orbranched lower alkenyl group having 2–6 carbon atoms or (d) a cycloalkylgroup having 3–8 carbon atoms (e.g., cyclopentyl, cyclohexyl,cycloheptyl and the like), R⁷ represents (a) a straight chain orbranched lower alkyl group having 1–6 carbon atom(s) (e.g., methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl,n-pentyl, isopentyl, neopentyl and the like), (b) a straight chain orbranched lower alkenyl group having 2–6 carbon atoms, (c) a lower alkylgroup having 1 to 3 carbon atom(s) substituted with a cycloalkyl grouphaving 3–8 carbon atoms (e.g., cyclopentyl, cyclohexyl, cycloheptyl andthe like) or an optionally substituted aryl group (e.g., a phenyl or anaphthyl group and the like, each of which may have a halogen atom,nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and the like) (e.g.,benzyl, p-chlorobenzyl, phenethyl, cyclopentylmethyl, cyclohexylmethyland the like), (d) a lower alkenyl group having 2 to 3 carbon atomssubstituted with a cycloalkyl having 3–8 carbon atoms or an optionallysubstituted aryl group (e.g., phenyl or naphthyl group and the like,each of which may have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower(C₁₋₄) alkoxy and the like) (e.g., a group having an alkenyl portionsuch as vinyl, propenyl, allyl, isopropenyl, and the like, for example,cinnamyl, and the like), (e) an optionally substituted aryl group (e.g.,a phenyl or naphthyl group and the like, each of which may have ahalogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄) alkoxy and thelike, such as phenyl, p-tolyl, naphthyl and the like), (f) a straightchain or branched lower alkoxy group having 1–6 carbon atom(s) (e.g.,methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy,t-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy and the like), (g) astraight chain or branched lower alkenyloxy group having 2 to 8 carbonatoms (e.g., allyloxy, isobutenyloxy and the like), (h) a cycloalkyloxygroup having 3–8 carbon atoms (e.g., cyclopentyloxy, cyclohexyloxy,cycloheptyloxy and the like), (i) a lower alkoxy group having 1 to 3carbon atom(s) substituted with a cycloalkyl having 3–8 carbon atoms(e.g., cyclopentyl, cyclohexyl, cycloheptyl and the like) or anoptionally substituted aryl group (e.g., a phenyl or naphthyl group andthe like, each of which may have a halogen atom, nitro, lower (C₁₋₄)alkyl, lower (C₁₋₄) alkoxy and the like) (e.g., a group having alkoxyportion(s) such as methoxy, ethoxy, n-propoxy, isopropoxy and the like,such as benzyloxy, phenethyloxy, cyclopentylmethoxy, cyclohexylmethoxyand the like), (j) a lower alkenyloxy group having 2 to 3 carbon atomssubstituted with a cycloalkyl having 3–8 carbon atoms (e.g.,cyclopentyl, cyclohexyl, cycloheptyl and the like) or an optionallysubstituted aryl group (e.g., a phenyl or naphthyl group and the like,each of which may have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower(C₁₋₄) alkoxy and the like) (e.g., a group having alkenyloxy portion(s)such as vinyloxy, propenyloxy, allyloxy, isopropenyloxy and the like,such as cinnamyloxy and the like) or (k) an optionally substitutedaryloxy group (e.g., phenoxy or naphthoxy group and the like, each ofwhich may have a halogen atom, nitro, lower (C₁₋₄) alkyl, lower (C₁₋₄)alkoxy and the like, such as phenoxy, p-nitro phenoxy, naphthoxy and thelike)]}, and the like], and the like.

As R², an optionally esterified carboxyl is preferred, and the specificexamples thereof include —COOH and a salt thereof, —COOMe, —COOEt,—COOtBu, —COOPr, pivaloyloxymethoxycarbonyl,1-(cyclohexyloxycarbonyloxy)ethoxycarbonyl,5-methyl-2-oxo-1,3-dioxolen-4-ylmethoxycarbonyl, acetoxymethoxycarbonyl,propionyloxymethoxycarbonyl, n-butyryloxymethoxycarbonyl,isobutyryloxymethoxycarbonyl, 1-(ethoxycarbonyloxy)ethoxycarbonyl,1-(acetoxy)ethoxycarbonyl, 1-(isobutyryloxy)ethoxycarbonyl,cyclohexylcarbonyloxymethoxycarbonyl, benzoyloxymethoxycarbonyl,cinnamyloxycarbonyl, cyclopentylcarbonyloxymethoxycarbonyl and the like,and may be any group as long as it is a group capable of forming ananion (e.g., COO⁻, its derivative, and the like) or a group which may beconverted into such a group under biological, i.e., physiologicalconditions (for example, an in vivo reaction such as oxidation,reduction or hydrolysis, and the like with an in vivo enzyme, and thelike), or chemically, or it may be carboxyl group or a prodrug thereof.

The above-mentioned R² is preferably the group represented by theformula —CO-D [wherein D represents (1) a hydroxy group or (2) lower(C₁₋₄) alkoxy in which the alkyl portion is optionally substituted witha hydroxy group, amino, halogen, lower (C₂₋₆) alkanoyloxy (e.g.,acetoxy, pivaloyloxy and the like), lower (C₃₋₈) cycloalkanoyloxy, lower(C₁₋₆) alkoxycarbonyloxy (e.g., methoxycarbonyloxy, ethoxycarbonyloxyand the like), lower (C₃₋₈) cycloalkoxycarbonyloxy (e.g.,cyclohexyloxycarbonyloxy and the like), lower (C₁₋₄) alkoxy or lower(C₃₋₈) cycloalkoxy]. Among these, carboxyl esterified with a lower(C₁₋₄) alkyl (preferably methyl or ethyl) is preferred.

In the above-mentioned formula, examples of the “hydrocarbon residue” ofthe “hydrocarbon residue which may link via a heteroatom and may besubstituted” represented by R³ include (1) an alkyl group, (2) analkenyl group, (3) an alkynyl group, (4) a cycloalkyl group, (5) an arylgroup, (6) an aralkyl group and the like. Among these, an alkyl group,an alkenyl group and a cycloalkyl group are preferred.

The alkyl group of the above-mentioned (1) may be any of straight chainor branched lower alkyl groups having about 1 to 8 carbon atom(s) suchas methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,t-butyl, pentyl, i-pentyl, hexyl, heptyl, octyl and the like.

The alkenyl group of the above-mentioned (2) may be any of straightchain or branched lower alkenyl groups having 2 to 8 carbon atoms suchas vinyl, propenyl, 2-butenyl, 3-butenyl, isobutenyl, 2-octenyl and thelike.

The alkynyl group of the above-mentioned (3) may be any of straightchain or branched lower alkynyl groups having 2 to 8 carbon atoms suchas ethynyl, 2-propynyl, 2-butynyl, 2-pentynyl, 2-octynyl and the like.

The cycloalkyl group of the above-mentioned (4) include lower cycloalkylhaving about 3 to 6 carbon atoms such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like.

Each of the above-mentioned alkyl group, alkenyl group, alkynyl group orcycloalkyl group may be substituted with a hydroxy group, an optionallysubstituted amino group (e.g., amino, N-lower (C₁₋₄) alkylamino,N,N-dilower (C₁₋₄) alkylamino and the like), halogen, a lower (C₁₋₄)alkoxy group, a lower (C₁₋₄) alkylthio group and the like.

The aryl group of the above-mentioned (5) includes, for example, phenyland the like, and the aralkyl group of the above-mentioned (6) include aphenyl-lower (C₁₋₄) alkyl and the like such as benzyl, phenethyl and thelike.

Each of the above-mentioned aralkyl group or aryl group may have, at thesubstitutable position(s) on the benzene ring, for example, halogen(e.g., F, Cl, Br and the like), nitro, an optionally substituted aminogroup (e.g., amino, N-lower (C₁₋₄) alkylamino, N,N-dilower (C₁₋₄)alkylamino and the like), lower (C₁₋₄) alkoxy (e.g., methoxy, ethoxy andthe like), lower (C₁₋₄) alkylthio (e.g., methylthio, ethylthio and thelike), lower (C₁₋₄) alkyl (e.g., methyl, ethyl and the like) and thelike.

Among the above-mentioned groups, as the “hydrocarbon residue” of the“hydrocarbon residue which may link via a heteroatom and may besubstituted” represented by R³, an optionally substituted alkyl oralkenyl group (e.g., lower (C₁₋₅) alkyl or lower (C₂₋₅) alkenyl groupand the like, each of which is optionally substituted with a hydroxygroup, an amino group, halogen or a lower (C₁₋₄) alkoxy group) arepreferred. Among these, a lower (C₁₋₅) alkyl (more preferably ethyl) ispreferred.

The “heteroatom” of the “hydrocarbon residue which may link via aheteroatom and may be substituted” represented by R³ include —O—,—S(O)_(m)— [m represents an integer of 0 to 2], —NR′— [R′ represents ahydrogen atom or lower (C₁₋₄) alkyl] and the like. Among these, —O— ispreferably used.

Among the above-mentioned groups, as R³, a lower (C₁₋₄) alkyl or lower(C₂₋₅) alkenyl group and the like, each of which may be linked via —O—,—S(O)_(m)— [m represents an integer of 0 to 2] or —NR′— [R′ represents ahydrogen atom or lower (C₁₋₄) alkyl] and may be substituted with asubstituent selected from a hydroxy group, an amino group, halogen andlower (C₁₋₄) alkoxy group, is preferred. Among these, a lower (C₁₋₅)alkyl or a lower (C₁₋₅) alkoxy (more preferably ethoxy) is preferred.

Among the compounds represented by the formula (I), abenzimidazol-7-carboxylic acid derivative represented by the formula(I′):

wherein R¹ is (1) a carboxyl group, (2) a tetrazolyl group or (3) agroup represented by the formula:

wherein i represents —O— or —S—, j represents >=O, >=S or >=S(O)_(m), mis as defined above, ring A represents a benzene ring which may befurther substituted with optionally substituted lower (C₁₋₄) alkyl(e.g., lower (C₁₋₄) alkyl optionally substituted with a hydroxy group, acarboxyl group, halogen and the like) or halogen and the like, inaddition to the substituent R² (preferably a benzene ring that does nothave any substituent except for R²), R² represents a group representedby the formula —CO-D [wherein D represents (1) a hydroxy group or (2)lower (C₁₋₄) alkoxy wherein the alkyl portion is optionally substitutedwith a hydroxy group, amino, halogen, lower (C₂₋₆) alkanoyloxy (e.g.,acetoxy, pivaloyloxy and the like), lower (C₃₋₈) cycloalkanoyloxy, lower(C₁₋₆) alkoxycarbonyloxy (e.g., methoxycarbonyloxy, ethoxycarbonyloxyand the like), lower (C₃₋₈) cycloalkoxycarbonyloxy (e.g.,cyclohexyloxycarbonyloxy and the like), lower (C₁₋₄) alkoxy or a lower(C₃₋₈) cycloalkoxy], R³ is a lower (C₁₋₅) alkyl or lower (C₂₋₅) alkenylgroup which may link via —O—, —S(O)_(m)— [m represents an integer of 0to 2] or —NR′— [R′ represents a hydrogen atom or lower (C₁₋₄) alkyl] andmay be substituted with a substituent selected from a hydroxy group, anamino group, halogen and a lower (C₁₋₄) alkoxy group (preferably lower(C₁₋₅) alkyl or lower (C₁₋₅) alkoxy; more preferably ethoxy), or apharmacologically acceptable salt thereof, is preferred. Among these,preferred are 2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylic acid [Candesartan],1-(cyclohexyloxycarbonyloxy)ethyl2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylate[Candesartan cilexetil], pivaloyloxymethyl2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylate,2-ethoxy-1-[[2′-(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl]benzimidazol-7-carboxylicacid or a salt thereof and the like.

The above-mentioned benzimidazol derivatives can be synthesized by, forexample, known methods disclosed in EP 425921, EP 459136, EP 553879, EP578125, EP 520423, EP 668272 and the like, or a similar manner thereto,and the like. Further, when Candesartan cilexetil is used, it ispreferable to use the stable C type crystal disclosed in EP 459136.

While the amount of the bioactive substance formulated into thesustained-release preparation of the present invention varies dependingon the kind of the bioactive substance and the like, it is generallyabout 0.1 to 50% (W/W), preferably about 0.2 to 30% (W/W), and morepreferably about 0.5 to 20% (W/W) in case of a bioactive peptide, or itis generally about 0.1 to 60% (W/W), preferably about 0.2 to 40% (W/W),and more preferably about 0.5 to 30% (W/W) in case of a non-peptidicbioactive substance.

The polymer used in the present invention is a polymer which is slightlysoluble or insoluble in water and has biocompatibility. Examples thereofinclude polystyrene, poly-acrylic acid, poly-methacrylic acid, acopolymer of acrylic acid and methacrylic acid, nylon, tetlon, siliconepolymer, dextran stearate, ethylcellulose, acetylcellulose,nitrocellulose, polyurethane, ethylene vinyl acetate copolymer,polyvinyl acetate, polyvinyl alcohol, polyacrylicamide and the like.Furthermore, examples of the biodegradable polymer include polymerssynthesized from one or more of α-hydroxycarboxylic acids (e.g.,glycolic acid, lactic acid and the like), hydroxydicarboxylic acids(e.g., malic acid and the like), hydroxytricarboxylic acids (e.g.,citric acid and the like) etc., by catalyst-free dehydrationpolycondensation, which have free carboxyl group(s), or a mixturethereof, poly-α-cyanoacrylic esters, polyamino acids (e.g.,poly-y-benzyl-L-glutamic acid and the like), maleic anhydride polymers(e.g., a styrene/maleic acid polymer and the like), and the like. Thepolymer may be a homopolymer or a copolymer. The type of polymerizationmay be of random, block or graft. When the above-mentionedα-hydroxycarboxylic acids, hydroxydicarboxylic acids andhydroxytricarboxylic acids have optically active centers in theirmolecular structures, they may be any of the D-, L- andDL-configurations.

Among these polymers, the biodegradable polymer having a free terminalcarboxyl group such as a polymer synthesized from α-hydroxycarboxylicacids (e.g., glycolic acid, lactic acid and the like) (e.g., poly-lacticacid, lactic acid/glycolic acid copolymer, and the like),poly-α-cyanoacrylic acid esters and the like are preferred.

The biodegradable polymer is more preferably a polymer synthesized fromα-hydroxycarboxylic acids and the like, especially preferably a lacticacid/glycolic acid polymer and the like.

Not only homopolymers such as poly-lactic acid, poly-glycolic acid,etc., but also lactic acid/glycolic acid copolymers are sometimes simplyreferred to as the lactic acid/glycolic acid polymer herein inclusively.

When the lactic acid/glycolic acid polymer (a lactic acid/glycolic acidcopolymer or homopolymer) is used as the biodegradable polymer, itscomposition ratio (mol %) is preferably about 100/0 to about 40/60, morepreferably about 85/15 to about 50/50.

The weight-average molecular weight of the above-described lacticacid/glycolic acid polymer is preferably about 3,000 to about 50,000,more preferably about 3,000 to about 25,000, further more preferablyabout 5,000 to about 20,000.

The degree of dispersion (weight-average molecular weight/number-averagemolecular weight) of the lactic acid/glycolic acid polymer is preferablyabout 1.2 to about 4.0, more preferably about 1.5 to about 3.5.

Regarding the weight-average molecular weight and the degree ofdispersion used herein, the former is a value converted into polystyreneas determined by gel permeation chromatography (GPC) using as referencesubstances 9 kinds of polystyrenes having the weight-average molecularweights of 120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and162, respectively, and the latter is calculated therefrom. The abovedetermination is carried out using GPC column KF804L×2 (manufactured byShowa Denko K. K.) and RI monitor L-3300 (manufactured by Hitachi Ltd.)with chloroform as a mobile phase.

Further, the biodegradable polymer having a free terminal carboxyl groupis a biodegradable polymer in which the number-average molecular weightbased on GPC measurement and the number-average molecular weight basedon terminal group quantification almost agree with each other. Thenumber-average molecular weight based on terminal group quantificationis calculated as follows:

About 1 to 3 g of the biodegradable polymer is dissolved in a mixedsolvent of acetone (25 ml) and methanol (5 ml), and the solution isquickly titrated with a 0.05 N alcoholic solution of potassium hydroxideusing phenolphthalein as an indicator while stirring at room temperature(20° C.) to determine the carboxyl group content; the number-averagemolecular weight based on terminal group quantification is calculatedfrom the following equation:Number-average molecular weight based on terminal groupquantification=20000×A/B

-   A: the mass (g) of the biodegradable polymer-   B: the amount (ml) of 0.05 N alcoholic solution of potassium    hydroxide added until the titration end point

The number-average molecular weight based on terminal groupquantification is an absolute value, while the number-average molecularweight based on GPC measurement is a relative value that variesdepending on various analytical conditions (e.g., kind of mobile phase,kind of column, reference substance, slice width chosen, baselinechosen, etc.). Therefore, although it is difficult to expressunequivocally and numerically, such description that “the number-averagemolecular weight based on GPC measurement and that based on terminalgroup quantification almost agree with each other” means, for example,that the number-average molecular weight based on terminal groupquantification of a polymer synthesized from α-hydroxycarboxylic acidsfalls within the range from about 0.5 to about 2 times, preferably fromabout 0.7 to about 1.5 times as much as the number-average molecularweight based on GPC measurement.

For example, in case of a polymer having a free terminal carboxyl groupand synthesized from one or more α-hydroxycarboxylic acids bycatalyst-free dehydration polycondensation, the number-average molecularweight based on GPC measurement and the number-average molecular weightbased on terminal group quantification almost agree with each other. Onthe other hand, in case of a polymer having substantially no freeterminal carboxyl group and synthesized from a cyclic dimer byring-opening polymerization using a catalyst, the number-averagemolecular weight based on terminal group quantification is significantlyhigher than (more than about 2 times) that based on GPC measurement.This difference makes it possible to clearly distinguish a polymerhaving a free terminal carboxyl group from a polymer having no freeterminal carboxyl group.

The lactic acid/glycolic acid polymer having a free terminal carboxylgroup can be produced by a per se known process, for example, thatdescribed in JP 61-28521 A (e.g., a process by a catalyst-freedehydration polycondensation reaction, or a dehydration polycondensationreaction in the presence of an inorganic solid acid catalyst, etc.).

While the decomposition/disappearance rate of the lactic acid/glycolicacid polymer varies widely, depending on the composition ratio or theweight-average molecular weight, the release duration can be extended(e.g., for about 6 months) by lowering the ratio of glycolic acid orincreasing the molecular weight, since decomposition/disappearance isgenerally delayed as the ratio of glycolic acid decreases. In contrast,the release duration can be shortened (e.g., for about one week) byincreasing the ratio of glycolic acid or decreasing the molecularweight. For obtaining a one week to two months type sustained-releasepreparation, it is preferable to use the lactic acid/glycolic acidpolymer whose composition ratio and weight-average molecular weight arewithin the above-described ranges.

Therefore, the composition of the biodegradable polymer used in thepresent invention is preferably selected according to the desired kindof a bioactive peptide and the desired duration of sustained-release.Specifically, for example, when GH is used as the bioactive peptide, alactic acid/glycolic acid polymer is preferably used. As the lacticacid/glycolic acid polymer, a preferred polymer is a lacticacid/glycolic acid copolymer having a lactic acid/glycolic acidcomposition ratio (mol %) of about 85/15 to about 50/50, more preferablyabout 75/25 to about 50/50. The weight-average molecular weight thereofis preferably about 8,000 to about 20,000, more preferably about 10,000to about 20,000. Further, the degree of dispersion (weight-averagemolecular weight/number-average molecular weight) of the lacticacid/glycolic acid polymer is about 1.2 to about 4.0, more preferablyabout 1.5 to about 3.5.

The lactic acid/glycolic acid polymer used can be produced by knownmethods such as those described in the above publications and the like.The polymer is preferably that produced by catalyst-free dehydrationpolycondensation. Any organic solvent used for the production of thepolymer and remaining in the polymer is removed after thepolymerization. As a method for this purpose, there are, for example,heat drying, vacuum drying, flash drying with dried gas, etc. However,such a solvent can be much more quickly removed by contacting withcontinuously provided high-pressure gas according to the presentinvention, thereby significantly reducing the time required for removingthe solvent. It is preferable to use the lactic acid/glycolic acidpolymer (PLGA) wherein the number-average molecular weight based onterminal group quantification and the number-average molecular weightbased on GPC measurement almost agree with each other.

Further, two kinds of lactic acid/glycolic acid polymers different inthe composition ratio and/or the weight-average molecular weight may beused by mixing them in an arbitrary ratio. An example thereof is amixture of a lactic acid/glycolic acid copolymer having the compositionratio of lactic acid/glycolic acid (mol %) of about 75/25 and theweight-average molecular weight of about 10,000, and a lacticacid/glycolic acid copolymer having the composition ratio of lacticacid/glycolic acid (mol %) of about 50/50 and the weight-averagemolecular weight is about 12,000. The preferred weight ratio of thesecopolymers upon mixing is about 25/75 to about 75/25.

The biodegradable polymer used in the present invention may be a metalsalt of the above mentioned biodegradable polymer. For example, therecan be used the various polyvalent metal salts of the biodegradablepolymer described in WO97/01331, and the like. Preferably, a polyvalentmetal salt of the lactic acid/glycolic acid polymer and the like, (morepreferably, zinc salt, calcium salt, magnesium salt and the like,further more preferably zinc salt and the like) can be used. The speciesof the metal of the polyvalent metal salt is not specifically limited aslong as it does not cause any adverse effect to a living body. Forexample, there can be used a polyvalent metal such as a divalent metal(e.g., iron, zinc, copper, calcium, magnesium, aluminum, tin, manganeseand the like), a trivalent metal (e.g., iron, aluminum, manganese andthe like), a tetravalent metal (e.g., tin and the like) and the like.

A metal salt of the biodegradable polymer is sometimes referred to asthe biodegradable polymer herein inclusively. For example, in case of apolyvalent metal salt of the lactic acid/glycolic acid polymer,sometimes, it is also referred to as the lactic acid/glycolic acidpolymer.

The above polyvalent metal salt of the biodegradable polymer can beproduced by the method described in WO97/01331 or similar methods.

In case that a polyvalent metal salt of the biodegradable polymer is azinc salt, it can be produced by reacting the biodegradable polymer withzinc oxide in an organic solvent.

In this method, first, a solution of the biodegradable polymer-zincoxide complex in an organic solvent is prepared by coexistence of thebiodegradable polymer with zinc oxide in the organic solvent. In thatcase, although the concentration of the biodegradable polymer in thesolvent varies depending on the molecular weight thereof, a kind of theorganic solvent and the like, for example, the concentration is about0.1 to about 80% (W/W), preferably about 1 to about 70% (W/W), morepreferably about 2 to about 60% (W/W). Further, although the amount ofzinc oxide to be added varies depending on the kind of the organicsolvent, for example, when the desired bioactive substance is a peptide,the amount is about 0.001 to about 2% (W/W), preferably about 0.01 toabout 1.5% (W/W), more preferably about 0.1 to about 1% (W/W), when thedesired bioactive substance is a non-peptide, the amount is about 0.001to about 30% (W/W), preferably about 0.01 to about 20% (W/W), morepreferably about 0.1 to about 10% (W/W), based on the amount of thebiodegradable polymer, as described in JP 10-231252 A.

Regarding the order of the addition of the biodegradable polymer andzinc oxide to the organic solvent, zinc oxide in the form of a powder orsuspended in the organic solvent can be added to a solution prepared bydissolving the biodegradable polymer in the organic solvent, or on thecontrary, a solution of the biodegradable polymer in the organic solventcan be added to a suspension prepared by suspending zinc oxide in theorganic solvent. Further, both of the biodegradable polymer and zincoxide can be mixed in the form of powders, then the organic solvent canbe added thereto. When the desired bioactive substance is a non-peptide,the organic solvent can be added after mixing of the biodegradablepolymer, zinc oxide and the bioactive substance in the form of powders.

The content of the biodegradable polymer in the preparation of thepresent invention is generally about 30 to 99.9% (W/W), preferably about60 to 97% (W/W), and more preferably about 70 to 90% (W/W).

The preparation of the present invention is produced by forming a solidmaterial containing the bioactive substance and the biodegradablepolymer and contacting the solid material with high-pressure gas.

The solid material containing the bioactive substance and thebiodegradable polymer is formed by, when the bioactive substance is abioactive peptide, for example, removing a solvent from a S/O dispersionobtained by dispersing a powder (S phase), which has been obtained bylyophilizing a solution of the bioactive peptide, in a solution of thebiodegradable polymer in an organic solvent (O phase), or removing asolvent from a W/O emulsion obtained by dispersing an aqueous phase (Wphase), which is an aqueous solution of the bioactive peptide, in asolution of the biodegradable polymer dissolved in an organic solvent,or removing a solvent from a solution of both bioactive peptide andbiodegradable polymer dissolved in an organic solvent (O phase). As amethod for this, there are, for example, (a) in-water drying method(S/O/W method and W/O/W or O/W method), (b) phase separation method(coacervation method) and (c) spray-drying method, or similar methodsthereto and the like. In the present description, the solid materialmeans a material in which constituents are linked to each otherphysically or chemically. The solid material includes, but notspecifically limited to, microcapsules and the like.

The organic solvent used for dissolving the biodegradable polymerpreferably has the boiling point of not lower than 30° C. Examples ofthe organic solvent includes halogenated hydrocarbons such asdichloromethane, dichloroethane, chloroform, carbon tetrachloride andthe like, aliphatic hydrocarbons such as pentane, hexane, heptane,cyclohexane, petroleum benzine, petroleum ether and the like, aromatichydrocarbons such as toluene, xylene and the like, alcohols such asmethyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol,benzyl alcohol and the like, polyhydric alcohols such as ethyleneglycol, propylene glycol and the like, esters such as methyl acetate,ethyl acetate and the like, organic acids such as formic acid, aceticacid, trifluoroacetic acid, trichloroacetic acid and the like, etherssuch as diethyl ether, isopropyl ether, dioxane, tetrahydrofuran and thelike, ketones such as acetone, methyl ethyl ketone and the like,nitrogen-containing compounds such as acetonitrile, propionitrile,pyridine, dimethylacetamide, dimethylformamide and the like,sulfur-containing compounds such as dimethylsulfoxide and the like, andthe like. These may be mixed in a suitable ratio. A solvent contained ina substance, especially an organic compound, e.g., a medicament shouldbe substantially removed from a product in view of properties of themedicament. Furthermore, for foods and general chemicals, residualsolvents in products are strictly regulated depending on theirapplication. The allowable amount of a residual solvent for medicamentsis described in the guideline based on the ICH (“A guideline forresidual solvent in medicament”, Pharm. Tech. Japan 16(5), 687–704,2000), and for example, the concentration limit for dichloromethane(classified into the Class 2) is 600 ppm, and the concentration limitfor acetone (classified into the Class 3) is 0.5% (5,000 ppm).

Moreover, an organic solvent used in dissolution of the biodegradablepolymer preferably has a boiling point of not higher than 120° C. Theorganic solvent includes such as halogenated hydrocarbons (e.g.,dichloromethane, chloroform and the like), alcohols (e.g., ethanol,methanol and the like), ethyl acetate, acetonitrile and the like. Thesemay be mixed in a suitable ratio. When an organic solvent is used alone,for example, dichloromethane, ethyl acetate, acetonitrile and the likeare preferred. When organic solvents are used as a mixed solvent, forexample, a combination of halogenated hydrocarbons (e.g.,dichloromethane, chloroform and the like) and alcohols (e.g., ethanol,methanol and the like) or acetonitrile is preferred. The mixing ratio(volume ratio) of the halogenated hydrocarbons and alcohols oracetonitrile is about 100:1 to about 1:1, and it is desirable to use amixed solvent having a mixing ratio of preferably about 30:1 to about2:1. Furthermore, while the concentration of the biodegradable polymerin a solution varies depending on the molecular weight, the kind of anorganic solvent, and the like, for example, it is about 0.01 to about80% (W/W), preferably about 0.1 to about 70% (W/W), and more preferablyabout 1 to about 60% (W/W).

Hereinafter, a method for microcapsulation, in case of the production ofsustained-release microcapsules as the preparation and using a bioactivepeptide as the bioactive substance, will be explained in detail.

(a-1) In-Water Drying Method (S/O/W Method)

According to this method, first, a water-miscible organic solvent and/ora volatile salt are added to an aqueous solution of the bioactivepeptide, and then, a bioactive peptide powder (S phase) is produced bylyophilization. A biodegradable polymer is dissolved in an organicsolvent, and then, the above bioactive peptide powder is dispersed intothe resulting organic solvent solution. The ratio (ratio by weight) ofthe bioactive peptide and the biodegradable polymer is, for example,about 1:1000 to about 1:1, preferably about 1:200 to about 1:5, morepreferably about 1:100 to about 1:5. Preferably, an external physicalenergy is applied to disperse the bioactive peptide powder uniformly inthe organic solvent solution. As a method for this, there can be used,for example, irradiation of ultrasonic wave, a turbine stirrer, ahomogenizer and the like. As to the average particle size of thebioactive peptide in the organic solvent solution, it is preferably notmore than about 10 μm, more preferably about 0.1 to 10 μm, further morepreferably about 0.5 to 5 μm. In the present invention, the averageparticle size of the bioactive peptide means the value obtained by usinga laser analytic particle size distribution measuring device (SALD2000A,manufactured by Shimadzu Corporation) after dispersing the bioactivepeptide in an organic solvent such as dichloromethane by using ahomogenizer. In this process, the bioactive peptide is added to theorganic solvent at the concentration of about 20 to 100 mg/ml, and thendispersed using a homogenizer, such as Polytron (manufactured byKinematica) at about 20,000 rpm for about 30 seconds to 1 minutes. Thedispersion is diluted appropriately with the organic solvent so that theaverage particle size can be measured with the above particle sizedistribution measuring device, followed by analysis.

Then, the organic solvent dispersion (S/O dispersion) thus prepared isadded to an aqueous solvent (W phase), and then the same externalphysical energy as that mentioned above, for example, irradiation ofultrasonic wave, a turbine stirrer, a homogenizer and the like isapplied to form a S/O/W emulsion. Then, the organic solvent of O phaseis evaporated to produce microcapsules. At this time, the volume of theaqueous phase is generally selected from about 1 times to about 10,000times, preferably about 2 times to about 5,000 times, more preferablyabout 5 times to about 2,000 times as much as the volume of the O phase.

An emulsifier can be added to the above external aqueous phase. As theemulsifier, there can be used any one which is generally capable offorming a stable S/O/W emulsion. Examples of the emulsifier includeanionic surfactants, nonionic surfactants, polyoxyethylene castor oilderivative, polyvinylpyrrolidones, polyvinyl alcohols,carboxymethylcelluloses, lecithin, gelatin, hyaluronic acids and thelike. These emulsifiers can be used by appropriately combining them. Theconcentration of the emulsifier in the external aqueous phase is,preferably about 0.001% to 20% (w/w), more preferably about 0.01% to 10%(w/w), particularly preferably about 0.05% to 5% (w/w).

The thus-obtained microcapsules are recovered by centrifugation orfiltration, washed with distilled water to remove the emulsifier and thelike adhering to the surface of microcapsules, re-dispersed in distilledwater, and lyophilized.

In the present invention, examples of the water-miscible organicsolvent, which can be added to the aqueous solution of the bioactivepeptide, include alcohols (e.g. methanol, ethanol, isopropanol and thelike, preferably methanol, ethanol and the like), acetone and the like.These may be used by mixing them at an appropriate ratio. Preferably, analcohol, more preferably ethanol is used alone. The amount(concentration) to be added to the aqueous solution of the bioactivepeptide is about 0.03 to 0.5% (V/V), preferably about 0.06 to 0.25%(V/V); more preferably about 0.1 to 0.15% (V/V), in terms ofvolume-ratio. By further lyophilizing the resultant aqueous solution ofthe bioactive peptide obtained by addition of the water-miscible organicsolvent, it is possible to prepare a bioactive peptide powder which iseasy to handle (superior operability) and is very fine (a small particlesize).

In the present invention, as the volatile salt, which is added to theaqueous solution of the bioactive peptide, there are, for example,ammonium salts (e.g., ammonium acetate, ammonium bicarbonate, ammoniumcarbonate, ammonium chloride and the like, preferably ammonium acetateand the like). The volatile salt can be used by mixing them at anappropriate ratio. The added amount of the volatile salt is about 10times to about 80 times mole, preferably about 10 times to about 70times mole, more preferably about 15 times to about 70 times mole,further more preferably about 20 times to about 70 times mole, mostpreferably about 20 times to about 50 times mole as much as the aqueoussolution of the bioactive peptide in terms of mole ratio. Bylyophilizing the resultant aqueous solution of the bioactive peptideobtained by addition of the volatile salt in a similar manner as theaddition of the water-miscible organic solvent, it is possible toprepare the bioactive peptide powder which is easy to handle (superioroperability) and is very fine (a small particle size).

In the present invention, the water-miscible organic solvent and/or thevolatile salt added to the aqueous solution of the bioactive peptide canbe used alone or in appropriate combination thereof. When thewater-miscible organic solvent and the volatile salt are used incombination thereof, they can be added to the aqueous solution of thebioactive peptide in accordance with the above amounts respectively.

(a-2) In-water Drying Method (W/O/W Method)

According to this method, water or a suitable buffer is added to thebioactive peptide to give a solution of the bioactive peptide (W phase).The biodegradable polymer is then dissolved in an organic solvent, andto this organic solvent solution is added the above-mentioned solutionof the bioactive peptide and the mixture is dispersed. The thus-obtainedW/O emulsion is added to an aqueous solvent (W phase). According to thesame method as the above-mentioned S/O/W method, microcapsules areobtained through a W/O/W emulsion.

(a-3) In-water Drying Method (O/W Method)

According to this method, the biodegradable polymer together with thebioactive peptide are dissolved in an organic solvent. The organicsolvent (O phase) is then added to an aqueous solvent (W phase).According to the same method as the above-mentioned S/O/W method,microcapsules are obtained through an O/W emulsion.

(b) Phase Separation Method (Coacervation Method)

According to this method, a coacervating agent is gradually added to theS/O dispersion of (a-1) or the W/O emulsion of (a-2) or the O phasesolution of (a-3) as described above with stirring to precipitate andsolidify microcapsules. The amount of the coacervating agent to be addedis about 0.01 to about 1,000 times by volume, preferably about 0.05 toabout 500 times by volume, especially preferably about 0.1 to about 200times by volume as much as the volume of the above dispersion. Anycoacervating agent can be used, as long as it is a polymeric, mineraloil or vegetable oil compound which is miscible with the organic solventused for dissolution of the biodegradable polymer but does not dissolvethe biodegradable polymer used. Specifically, examples of thecoacervating agent include silicone oil, sesame oil, soybean oil, cornoil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane,n-heptane and the like. Two or more of these can be used in combination.The thus-obtained microcapsules are recovered by filtration, washedrepeatedly with heptane and the like to remove the coacervating agent.Further, washing is carried out in the same manner as that in the above(a), followed by lyophilization.

In the production of microcapsules by the in-water drying method orcoacervation method, an antiaggregation agent can be added forpreventing aggregation of particles. Examples of the antiaggregationagent can be used, for example, water-soluble polysaccharides such asmannitol, lactose, glucose, starches (e.g., corn starch and the like),hyaluronic acid and its alakaline metal salt, etc.; protein such asglycine, fibrin, collagen, etc.; inorganic salts such as sodiumchloride, sodium hydrogen phosphate, etc.; and the like.

(c) Spray-Drying Method

In this method, microcapsules are produced by spraying the S/Odispersion of (a-1), the W/O emulsion of (a-2) or the O phase solutionof (a-3) described above via a nozzle into the drying chamber of a spraydrier to volatilize the organic solvent in fine droplets within a veryshort time. As the nozzle, there are, for example, a two-fluid nozzletype, a pressure nozzle type and a rotary disc type and the like. It isalso advantageous, if necessary, to spray an aqueous solution of theabove-described antiaggregation agent via another nozzle in order toprevent aggregation of microcapsule particles.

The solid material formed by the above-mentioned method such asmicrocapsules containing the bioactive substance and the biodegradablepolymer, and the like, is then contacted with high-pressure gas(preferably carbon dioxide) to further extract and remove the organicsolvent.

Specifically, for example, a lyophilized microcapsule powder obtained by(a) is fed into an extraction vessel, and extraction treatment iscarried out with an extraction system comprising a carbon dioxidedelivery pump and a pressure regulating valve. Alternatively, amicrocapsule suspension before lyophilization, obtained by (a) or (b)may be fed into an extraction vessel and subjected to extractiontreatment similarly. In these cases, the extraction treatment isdesirably carried out under more gentle conditions so as to notdeteriorate the quality of a sustained-release preparation.

The high-pressure gas in the present invention is gas at pressure notless than the atmospheric pressure at a given temperature but not morethan the liquefying pressure at said temperature.

Examples of the high-pressure gas used in the present invention includecarbon dioxide, nitrous oxide, nitrogen, helium, argon, alkane (e.g.,ethane, propane and the like), alkene (e.g., ethylene and the like), andthe like. While these may be used by mixing them in a suitable ratio,preferably, it is desirable to use carbon dioxide alone.

When a temperature of high-pressure gas contacting with a preparation ismuch higher than the glass transition temperature of a biodegradablepolymer used as a substrate of the preparation, the risk of adhesion,deformation, decomposition of the bioactive substance, deterioration andthe like of the preparation increases. The glass transition temperaturein the present invention means medium glass transition temperatureobtained by rising temperature at the rate of 10 or 20° C./min using adifferential scanning calorimeter (DSC). Alternatively, when atemperature of high-pressure gas is too low, the removal of an organicsolvent becomes insufficient. The organic solvent is preferably removedto less than 1,000 ppm, preferably less than 500 ppm, and morepreferably less than 100 ppm. Therefore, the advantageous temperaturefor using carbon dioxide as high-pressure gas in the present inventionis within a temperature range of +20 to −60° C., preferably +10 to −50°C., more preferably 0 to −40° C., still more preferably −5 to −30° C.,and the most preferably −10 to −25° C., based on the glass transitiontemperature of the biodegradable polymer (generally about 20 to 60° C.).

While the range of pressure varies depending on the selectedhigh-pressure gas, but generally, when the pressure of high-pressure gasis too high, the risk of adhesion, deformation, increase of the initialrelease immediately after administration and the like for themicrocapsules increases, or when the pressure is too low, the removal ofthe organic solvent becomes insufficient. The advantageous pressure forusing carbon dioxide as high-pressure gas in the present inventionpressure is about 1 to 7 MPa, preferably about 1 to 4 MPa, and morepreferably about 2 to 4 MPa.

While the period for contacting with the high-pressure gas variesdepending on the pressure of the high-pressure gas, temperature, theamount of microcapsules to be treated and the like, it is preferablyabout 5 minutes to about 48 hours when carbon dioxide is used ashigh-pressure gas. More preferably, it is about 10 minutes to about 12hours.

Hereinafter the step for high-pressure gas treatment of microcapsulesusing carbon dioxide in a high-pressure gaseous state will be explainedin more detail with referring to FIG. 1. FIG. 1 is a schematic drawing,which exemplifies an apparatus used for the high-pressure gas treatmentin the present invention. Such apparatus for high-pressure gas treatmentcomprises, for example, as shown in FIG. 1, a liquefied carbon dioxidebomb 1, a carbon dioxide delivery pump 2, a heat exchanger 3, anextraction vessel 4, a thermostat 5, a detector 6, an automaticpressure-regulating valve 7 and a recovery vessel 8. Microcapsules to betreated are fed into the extraction vessel 4, and the apparatus issealed and heated to a predetermined temperature. The liquefied carbondioxide is then delivered from the liquefied carbon dioxide bomb 1 tothe heat exchanger 3 by the carbon dioxide delivery pump 2, heated to apredetermined temperature, and converted into a high-pressure gaseousstate. The carbon dioxide in the high-pressure gaseous state is thenblown into the extraction vessel 4 to dissolve and extract the solventin the microcapsules into the high-pressure gas. The extracted solventis recovered in the recovery vessel 8 via the detector 6 and automaticpressure regulating valve 7. The pressure applied to the whole system iscontrolled by the automatic pressure regulating valve 7 connected to thelowest downstream. By contacting with the high-pressure gas for a givenperiod, the excess amount of the initial release of the bioactivesubstance immediately after administration is markedly suppressed, andthe residual organic solvent can be removed without producingaggregates, related substances or reactants of the bioactive peptide.

The sustained-release preparation of the present invention is preferablyin the form of fine particles. That is, the sustained-releasepreparation does not provide undue pain to a patient, when it isadministered to said patient using an injection needle, which isgenerally used for subcutaneous or intramuscular injection. The particlesize of the sustained-release preparation is, for example, about 0.1 to300 μm, preferably about 1 to 150 μm, specifically preferably about 2 to100 μm in terms of a mean particle diameter. The content of thebioactive substance contained in the sustained-release preparation ofthe present invention is, for example, in case of a bioactive peptide,generally about 0.1 to 50% (W/W), preferably about 0.2 to 30% (W/W), andmore preferably about 0.5 to 20% (W/W). The content of the biodegradablepolymer contained in the sustained-release preparation of the presentinvention is generally about 30 to 99.9% (W/W), preferably about 60 to97% (W/W), and more preferably about 70 to 90% (W/W).

The initial release percentage of the sustained-release preparation ofthe present invention [the release percentage up to one day (24 hours)after administration] is, in case of a bioactive peptide, preferablyabout not more than 40%, more preferably about 1 to 40%, and morepreferably about 3 to 35%.

The sustained-release preparation of the present invention can beadministered as microcapsules or as preparations in various formsprepared by using microcapsules as a raw material, such as parenteralpreparations (e.g., injectable preparations or preparations forimplantation in muscle, subcutaneous, organs and the like, preparationsfor administering to mucosa onto cavitas nasi, rectum, uterus, etc.),oral preparations (e.g., capsules (hard capsules, soft capsules, etc.),solid preparations such as granules and powders, etc., liquidpreparations such as suspensions, etc.), and the like.

In particular, the sustained-release preparation of the presentinvention is preferably for injection. For example, in case that thesustained-release preparation is microcapsules, it is possible to obtaina practical sustained-release preparation for injection by formulatingthe microcapsules in an aqueous suspension together with a dispersingagent (e.g., a surfactant such as Tween 80, HCO-60, etc.,polysaccharides such as carboxymethyl celluloses, sodium alginate,hyaluronic acid, etc.), a preservative (e.g., methylparaben,propylparaben, etc.), a tonicity agent (e.g., sodium chloride, mannitol,sorbitol, glucose, etc.), and the like. It is also possible to obtain apractical sustained-release preparation for injection by dispersing themicrocapsules together with a vegetable oil such as sesame oil, cornoil, etc., or a mixture thereof with a phospholipid such as lecithin, ora medium-chain fatty acid triglyceride (e.g., Miglyol 812) to prepare anoily suspension.

When the sustained-release preparation is, for example, microcapsules,the particle size of the sustained-release preparation for an injectablesuspension can be selected from the range satisfying the requirementsfor the degree of dispersion and the needle passability for theinjection. For example, the particle size is within the range of about0.1 to about 300 μm, preferably about 1 to about 150 μm, more preferablyabout 2 to about 100 μm, as the average particle size.

Methods for producing a sterile preparation from the above microcapsulesinclude, but are not limited to, to carry out entire production stepsaseptically, to sterilize with gamma rays, to add an antiseptic, and thelike.

The sustained-release preparation can be safely used in mammals (e.g.,human, cattle, pig, dog, cat, mouse, rat, rabbit and the like) with lowtoxicity.

Indication of the sustained-release preparation varies depending on abioactive peptide used. The sustained-release preparation is useful toprevent or treat diabetes when insulin is used as the bioactive peptide;viral hepatitis (e.g., type C hepatitis, HBe antigen-positive activehepatitis and the like) and cancer (e.g., renal carcinoma, multiplemyeloma and the like) when interferon-α is used; anemia (e.g., anemiaduring dialysis of kidney and the like) when erythropoietin is used;neutropenia (e.g., in cancer therapy and the like) and infections whenG-CSF is used; cancer (e.g., hemangioendothelioma and the like) whenIL-2 is used; fracture, wound (e.g., bedsore and the like),periodontitis and gastrointestinal ulcer when FGF is used;thrombocytopenia when FGF-9 is used; senile dementia and neuropathy whenNGF is used; thrombosis when TPA is used; and cancer when tumor necrosisfactor is used. Further, the sustained-release preparation containing GHis applied to Turner's syndrome, chronic renal diseases, achondroplasia,and adult hypopituitarisin (adult GHD), in addition to pituitarydwarfism, based on growth hormone activity of GH. Further, since, GH isreported to be applied to diseases such as Down syndrome, Silversyndrome, hypochondroplasia and juvenile chronic arthritis to provideexcellent therapeutic effects, the sustained-release preparationcontaining GH can also be applied to these diseases. Thesustained-release preparation containing GH is also useful to prevent ortreat congestive heart-failure and the like. The other indications towhich the sustained-release preparation containing GH can be appliedinclude, hematogenesis during organ implantation or treatment for apatient suffering from AIDS with a drug, improvement ofhypoalimentation, renal anemia, angina pectoris, hyperlipidemia,obesity, acceleration of treatment for burn, wound or ulcer,invasiveness from surgery (operation, lesion), early recovery afteroperation, sepsis, prevention of fracture due to osteoporosis, earlyrecovery of muscular strength of a patient suffering from fracture dueto osteoporosis, amyotropic lateral scelosis (ALS), decubitus and thelike. Furthermore, it is expected to have effects as an antiaging agentaiming at improving the quality of life (QOL) for frail aged persons, oreffects for suppressing the development or improving neurodegenerativediseases (Alzheimer's disease, Parkinson's disease, cerebrovasculardisease and the like) due to the nerve protective effect of hGH. Byforming GH into a sustained-release preparation, drug effects superiorto those of a GH subcutaneous injection can be obtained for theseindications. When the bioactive substance is candesartan, thepreparation is effective for the prevention or improvement ofcardiomegaly, cardiac failure, myocardial infarct, cerebral stroke,ischemic peripheral neuropathy, myocardial ischemia, venousincompetence, development of cardiac failure after myocardial infarct,diabetic nephropathy, nephritis, glomerular nephritis, arteriosclerosis,vascular hypertrophy, vascular hypertrophy or occulusion afterpercutaneous transluminal coronary angioplasty, vascular re-occulusionafter bypass operation, hyperaldosteronism, glomerular sclerosis, renalfailure, glaucoma, ocular hypertension, hyperlipidemia, stenocardia,aneurysm, coronary arteriosclerosis, cerebral arteriosclerosis,peripheral arteriosclerosis, thrombosis, central nerve system disease,Alzheimer's disease, amnesia, depression, amnestic syndrome, seniledementia, dysesthesia, multiple organ failure, prevention or treatmentof disease or sclerodermia associated with endothelial disorder, orsymptom of anxiety, symptom of strain, unpleasantmental state ormaldigestion.

Although the dose of the sustained-release preparation varies dependingon a particular kind and amount of the bioactive peptide, releaseduration, target disease, subject animal species and other factors, itcan be set at any level, as long as an effective concentration of thebioactive peptide in the body is maintained. For example, when thesustained-release preparation is one designed for two week release, thedose of the bioactive peptide can be suitably chosen from the range ofpreferably about 0.0001 to about 10 mg/kg body weight, more preferablyabout 0.05 to about 3 mg/kg body weight, per an adult. The preferredadministration frequency of the sustained-release preparation can besuitably chosen from once a week, once every two weeks, once a month,once every two months and the like, depending on a particular kind andamount of the bioactive peptide, dosage form, release duration, targetdisease, subject animal species and other factors. Preferably, thesustained-release preparation includes a one week to two months typesustained-release preparation, more preferably one week to one monthtype sustained-release preparation.

When the bioactive peptide as an active component in thesustained-release preparation is, for example, insulin, the dose peradministration to an diabetic adult is suitably chosen from the range ofusually about 0.001 to about 1 mg/kg body weight, preferably about 0.01to about 0.2 mg/kg body weight, as an effective ingredient. And thepreferred administration frequency is once a week.

When the bioactive peptide as an active component in thesustained-release preparation is GH, the dose can be set at any level,as long as an effective concentration of GH in the body is maintained,although varying depending on a particular kind and amount of GH,release duration, target disease, subject animal species and otherfactors. Regarding the treatment of the above described diseases, whenthe sustained-release preparation is one designed for two week release,the dose of GH can be suitably chosen from the range of about 0.01 toabout 5 mg/kg body weight (about 0.03 to about 15 IU/kg body weight),more preferably about 0.05 to about 1 mg/kg body weight (about 0.15 toabout 3 IU/kg body weight), per a child or an adult for safeadministration. The preferred administration frequency can be suitablychosen from once a week, once every two weeks, once a month and etc.,depending on a particular amount of GH, dosage form, release duration,target disease, subject animal species and other factors, preferably oneweek to two months-type sustained-release preparation, more preferablyone week to one month-type sustained-release preparation.

The sustained-release preparation is preferably stored at ordinarytemperature or in a cold place. More preferably, the sustained-releasepreparation is stored in a cold place. The “ordinary temperature” andthe “cold place” are defined in the Pharmacopoeia of Japan. Namely, the“ordinary temperature” means 15 to 25° C., and the “cold place” means atemperature of not more than 15° C. In the “cold place”, it is morepreferably about 2 to 8° C.

Hereinafter the present invention will be explained more specificallywith referring to the Reference Examples, Examples and Test Examples,which do not limit the present invention.

REFERENCE EXAMPLE 1

To an aqueous solution of gene recombinant hGH (final hGHconcentration=2 mg/ml) was added ammonium acetate (20-fold molequivalent). The mixture (100 ml) was dropwise added to the inner wallsurface of a distillation flask cooled in a dry ice-ethanol bath using aperistaltic pump over 30 minutes to rapid-freeze the mixture and thefrozen mixture was dried in vacuo to obtain hGH powder. A lacticacid-glycolic acid copolymer (lactic acid/glycolic acid=65/35,viscosity=0.160 dl/g, 1.690 g) and zinc oxide (10 mg) were dissolved indichloromethane (2.7 ml). To the organic solvent solution was added theabove-mentioned hGH powder (359 mg) and the mixture was finelygranulated with Polytron (manufactured by Kinematica). This S/Odispersion was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) and the mixture was stirred and emulsified using a homomixer.The mixture was stirred at room temperature for 3 hours to volatilizedichloromethane and centrifuged (about 1,500 rpm) to obtainedmicrocapsules. The microcapsules were then washed twice with distilledwater (400 ml) and lyophilized from D-mannitol (0.2 g) to obtainlyophilized hGH-containing microcapsule powder. Under the sameconditions, six batches of the microcapsules were produced. The yield ofthe lyophilized microcapsule powder obtained was 6.8 g.

REFERENCE EXAMPLE 2

To an aqueous solution of gene recombinant hGH (final hGHconcentration=2 mg/ml) was added ammonium acetate (20-fold molequivalent). A lactic acid-glycolic acid copolymer (lactic acid/glycolicacid=50/50, viscosity=0.154 dl/g, 1.850 g) and zinc oxide (10 mg) weredissolved in dichloromethane (2.7 ml). To the organic solvent solutionwas added the above-mentioned hGH powder (155 mg) and the mixture wasfinely granulated with Polytron (manufactured by Kinematica). This S/Odispersion was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) and the mixture was stirred and emulsified using a homomixer.The mixture was stirred at room temperature for 3 hours to volatilizedichloromethane and centrifuged (about 1,500 rpm) to obtainmicrocapsules. The microcapsules were then washed twice with distilledwater (400 ml) and lyophilized from D-mannitol (0.2 g) to obtainlyophilized hGH-containing microcapsule powder. Under the sameconditions, six batches of the microcapsules were produced. The yield ofthe lyophilized microcapsule powder obtained was 7.6 g.

REFERENCE EXAMPLE 3

To an aqueous solution of gene recombinant hGH (final hGHconcentration=2 mg/ml) was added ammonium acetate (20-fold molequivalent). The mixture (100 ml) was dropwise added to the inner wallsurface of a distillation flask cooled in a dry ice-ethanol bath using aperistaltic pump over 30 minutes so as to rapid-freeze the mixture andthe frozen mixture was dried in vacuo to obtain hGH powder. A lacticacid-glycolic acid copolymer (lactic acid/glycolic acid=65/35,viscosity=0.160 dl/g, 1.521 g) and zinc oxide (9 mg) were dissolved indichloromethane (2.4 ml). To the organic solvent solution was added theabove-mentioned hGH powder (270 mg) and the mixture was finelygranulated with Polytron (manufactured by Kinematica). This S/Odispersion was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) that had been cooled to 18° C., and the mixture was stirred andemulsified using a homomixer. The mixture was stirred at roomtemperature for 3 hours to volatilize dichloromethane and centrifuged(about 1,500 rpm) to obtain microcapsules. To the microcapsulesuspension, which had been obtained by removing the supernatant as muchas possible by aspiration operation, was added a 50% aqueous solution ofethanol (500 ml), and the mixture was stirred gently using a propellerat room temperature for 15 minutes. The mixture was centrifuged (about1,500 rpm) to obtain microcapsules. The microcapsules were then washedtwice with distilled water (400 ml) and lyophilized from D-mannitol (180mg) to give lyophilized hGH-containing microcapsule powder. In order toremove the residual solvent, the powder was dried in vacuo at 46° C. for72 hours to obtain microcapsules.

REFERENCE EXAMPLE 4

Evaluation of Pharmacological Effect for Human Growth Hormone-containingMicrocapsules

To female SD rats, which had been removed glandula pituitaria atfour-week old, was administered an immunosuppressive agent, tacrolimus(Prograf injection, manufactured by Fujisawa Pharmaceutical Co., Ltd.)to suppress the production of antibodies to hGH. Microcapsules wereadministered to the animal at six-week old, and the body weight, bodylength and concentration of rat insulin-like growth factor I (rIGF-I) inblood serum were measured for 5 weeks. Specifically, the Prografinjection (5 mg) was diluted with saline, and the dilution was injectedsubcutaneously, at the dose of 50 μg/0.2 ml/rat at three days before thefirst administration of microcapsules, immediately after the firstadministration of microcapsules and on the 4th, 7th and 11th days afterthe first administration, and at the dose of 75 μg/0.2 ml/rat on the14th, 18th, 21st, 25th, 28th and 32nd days after the firstadministration, respectively. Furthermore, in order to more physicallynormalize the glandula pituitaria-removed rat, hormone supplementationwas also carried out. A mixed solution of sodium L-thyroxin pentahydrateand hydrocortisone succinate (both are manufactured by Wako purechemical Industries, Ltd) (the final concentrations were 1 μg and 50 μgper 0.2 ml/rat, respectively) was subcutaneously administered threetimes a week, namely, three days before the first administration of themicrocapsules, immediately after the first administration, and on the2nd, 4th, 7th, 9th, 11th, 14th, 16th, 18th, 21st, 23rd, 25th, 28th, 30thand 32nd days after the first administration. The microcapsules weredispersed in a dispersion medium (5% mannitol, 0.5%carboxymethylcellulose sodium, 0.1% Tween 80) so as to be 24 mg hGH/ml,and 0.5 ml of the dispersion was administered subcutaneously to the backof the rat under ether anesthesia. The dose was 12 mg as hGH. After theadministration of microcapsules, the body weight and body length of therat was measured with time up to 35 days. In addition, blood wascollected from the caudal vein with time and blood serum wasfractionated. The concentration of rIGF-I in blood serum was measured byradioimmunoassay (DSL-2900, Diagnostic Systems Laboratories, Inc.).

REFERENCE EXAMPLE 5

Candesartan (2.0 g), zinc oxide (manufactured by Hakusui ChemicalIndustries, Ltd., 0.37 g) and a lactic acid-glycolic acid copolymer(lactic acid/glycolic acid 75/25 (mol %), weight-average molecularweight 8,700, manufactured by Wako Pure Chemical Industries, Ltd, 3.6 g)were added to a mixed solution of dichloromethane (12.75 ml), methanol(2.25 ml) and acetic acid (0.136 ml), and the mixture was stirred withshaking at room temperature overnight to obtain a homogenous solution.The solution was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) containing 20 mM of zinc acetate, which had been previouslyadjusted to 18° C., and an O/W emulsion was prepared using a turbinetype homomixer at 7,000 rpm. This O/W emulsion was stirred at roomtemperature for 3 hours to volatilize dichloromethane, methanol andacetic acid, and the oil phase was solidified and collected usingcentrifuge at 3,000 rpm. This was dispersed in distilled water again andfurther centrifuged to wash out free drug and the like. The collectedmicrocapsules were dispersed again by adding distilled water containingmannitol (0.8 g) and lyophilized to obtain a powder. The encapsulationratio of candesartan in microcapsules was 90.9%, and the content ofcandesartan in microcapsules/mannitol powder was 26.5%.

REFERENCE EXAMPLE 6

One batch was conducted in the following amount for treatment.Candesartan (2.0 g), zinc oxide (manufactured by Hakusui ChemicalIndustries, Ltd., 0.37 g) and a lactic acid-glycolic acid copolymer(lactic acid/glycolic acid 75/25 (mol %), weight-average molecularweight 8,700, manufactured by Wako Pure Chemical Industries, Ltd, 3.6 g)were added to a mixed solution of dichloromethane (12.75 ml), methanol(2.25 ml) and acetic acid (0.136 ml), and the mixture was stirred withshaking at room temperature overnight to obtain a homogenous solution.The solution was added to a 0.1% aqueous solution of polyvinyl alcohol(800 ml) containing 10 mM of zinc acetate, which had been previouslyadjusted to 18° C., and an O/W emulsion was prepared using a turbinetype homomixer at 7,000 rpm. This O/W emulsion was stirred at roomtemperature for 3 hours to volatilize dichloromethane, methanol andacetic acid, and the oil phase was solidified and collected usingcentrifuge at 3,000 rpm. This was dispersed in distilled water again andfurther centrifuged to wash out free drug and the like. Theabove-mentioned operations were conducted by two batches, and themicrocapsules of the two batches were mixed, and the microcapsules weredispersed again by adding distilled water containing mannitol (1.6 g)and lyophilized to obtain a powder. The encapsulation ratio ofcandesartan in microcapsules was 90.7%, and the content of candesartanin microcapsules/mannitol powder was 26.4%.

EXAMPLE 1

The solvent was removed under the following four conditions using 0.3 gof the hGH-containing lyophilized microcapsule powder obtained inReference Example 1, respectively. The microcapsule powder wastransferred into an extraction vessel (volume 10 ml) of a supercriticalfluid extraction apparatus (manufactured by JASCO Corporation). Theapparatus was sealed and heated to a predetermined temperature in athermostat. Carbon dioxide was delivered to a heat exchanger via adelivery pump (SCF-Get) at the bomb pressure (about 6 to 7 MPa) andheated to the given temperature. The pressure applied to the wholesystem was controlled by an automatic pressure regulating valve(SCF-Bpg), and the carbon dioxide was converted into a high-pressuregaseous state at given pressure. The high-pressurized carbon dioxide gaswas then blown into an extraction vessel, and the solvent was removedunder the following four conditions.

-   (1) Pressure 2 MPa, temperature 15° C., extraction period 15    minutes.-   (2) Pressure 2 MPa, temperature 15° C., extraction period 30    minutes.-   (3) Pressure 2 MPa, temperature 15° C., extraction period 45    minutes.-   (4) Pressure 2 MPa, temperature 15° C., extraction period 60    minutes.

EXAMPLE 2

The solvent was removed under the following four conditions using 0.3 gof the hGH-containing lyophilized microcapsule powder obtained inReference Example 2, respectively. The microcapsule powder wastransferred into an extraction vessel (volume 10 ml) of a supercriticalfluid extraction apparatus (manufactured by JASCO Corporation). Theapparatus was sealed and heated to a given temperature in a thermostat.Carbon dioxide was delivered to a heat exchanger via a delivery pump(SCF-Get) at the bomb pressure (about 6 to 7 MPa) and heated to thepredetermined temperature. The pressure applied to the whole system wascontrolled by an automatic pressure regulating valve (SCF-Bpg), and thecarbon dioxide was converted into a high-pressure gaseous state atpredetermined pressure. The high-pressurized carbon dioxide gas was thenblown into an extraction vessel, and the solvent was removed under thefollowing four conditions.

-   (1) Pressure 2 MPa, temperature 15° C., extraction period 30    minutes.-   (2) Pressure 2 MPa, temperature 15° C., extraction period 60    minutes.-   (3) Pressure 2 MPa, temperature 15° C., extraction period 180    minutes.-   (4) Pressure 1 MPa, temperature 150° C., extraction period 720    minutes.

EXAMPLE 3

The solvent was removed under the following 18 conditions using 0.3 g ofthe candesartan-containing lyophilized microcapsule powder obtained inReference Example 5, respectively. The microcapsule powder wastransferred into an extraction vessel (volume 10 ml) of a supercriticalfluid extraction apparatus (manufactured by JASCO Corporation). Theapparatus was sealed and heated to a predetermined temperature using athermostat. Carbon dioxide was delivered to a heat exchanger via adelivery pump (SCF-Get) at the bomb pressure (about 6 to 7 MPa) andheated to the given temperature. The pressure applied to the wholesystem was controlled by an automatic pressure regulating valve(SCF-Bpg), and the carbon dioxide was converted into a high-pressuregaseous at the given pressure. The high-pressurized carbon dioxide gaswas then blown into an extraction vessel, and the solvent was removedunder the following 18 conditions.

-   (1) Pressure 2.0 MPa, temperature 15° C., extraction period 30    minutes.-   (2) Pressure 2.0 MPa, temperature 15° C., extraction period 60    minutes.-   (3) Pressure 2.0 MPa, temperature 15° C., extraction period 120    minutes.-   (4) Pressure 2.0 MPa, temperature 15° C., extraction period 180    minutes.-   (5) Pressure 2.5 MPa, temperature 15° C., extraction period 30    minutes.-   (6) Pressure 2.5 MPa, temperature 15° C., extraction period 60    minutes.-   (7) Pressure 2.5 MPa, temperature 15° C., extraction period 120    minutes.-   (8) Pressure 2.5 MPa, temperature 15° C., extraction period 180    minutes.-   (9) Pressure 3.0 MPa, temperature 15° C., extraction period 15    minutes.-   (10) Pressure 3.0 MPa, temperature 15° C., extraction period 30    minutes.-   (11) Pressure 3.0 MPa, temperature 15° C., extraction period 60    minutes.-   (12) Pressure 3.0 MPa, temperature 15° C., extraction period 120    minutes.-   (13) Pressure 3.0 MPa, temperature 15° C., extraction period 180    minutes.-   (14) Pressure 3.5 MPa, temperature 15° C., extraction period 30    minutes.-   (15) Pressure 3.5 MPa, temperature 15° C., extraction period 60    minutes.-   (16) Pressure 3.5 MPa, temperature 15° C., extraction period 120    minutes.-   (17) Pressure 3.5 MPa, temperature 15° C., extraction period 180    minutes.-   (18) Pressure 4.0 MPa, temperature 15° C., extraction period 30    minutes.

EXAMPLE 4

The solvent was removed under the following three conditions using thecandesartan-containing lyophilized microcapsule powder obtained inReference Example 6, respectively. The microcapsule powder wastransferred into an extraction vessel (volume 10 ml) of a supercriticalfluid extraction apparatus (manufactured by JASCO Corporation). Theapparatus was sealed and heated to a predetermined temperature using athermostat. Carbon dioxide was delivered to a heat exchanger via adelivery pump (SCF-Get) at the bomb pressure (about 6 to 7 MPa) andheated to a given temperature. The pressure applied to the whole systemwas controlled by an automatic pressure regulating valve (SCF-Bpg), andthe carbon dioxide was converted into a high-pressure gaseous state at agiven pressure. The high-pressurized carbon dioxide gas was then blowninto an extraction vessel, and the solvent was removed under thefollowing three conditions.

-   (1) pressure 3.0 MPa, temperature 15° C., extraction period 60    minutes, charged amount of the microcapsules 0.3 g.-   (2) pressure 3.0 MPa, temperature 15° C., extraction period 60    minutes, charged amount of the microcapsules 2 g.-   (3) pressure 3.0 MPa, temperature 15° C., extraction period 60    minutes, charged amount of the microcapsules 5 g.

TEST EXAMPLE 1

For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 1 (1) to (4), the amount of residualdichloromethane (DCM) and hGH content in the microcapsules were measuredby the following method.

(1) Amount of Residual Dichloromethane (DCM)

Microcapsules (about 100 mg) were weighed precisely, dissolved indimethylsulfoxide and made up to exactly 5 ml to prepare a samplesolution. Separately, dichloromethane (about 1 g) was weighed preciselyand made up to exactly 20 ml with addition of dimethylsulfoxide. Thissolution was diluted by exactly 10000 times with dimethylsulfoxide toobtain a standardized solution. The sample solution and standardizedsolution (each 1 μl) were tested by gas chromatography under thefollowing conditions, and the peak area of dichloromethane for eachsolution was measured by automatic integration to calculate the amountof dichloromethane.

-   Detector: hydrogen flame ionization detector-   Column: OVI-G43 film thickness 3 μm, 0.53 mm i.d.×30 m (Supelco)-   Inlet temperature: 140° C.-   Detector temperature: 260° C.-   Column temperature: 40° C. (10 min retention)→240° C. (35°    C./min)→240° C. (20 min retention)→cooling→40° C.-   Carrier gas: helium-   Flow: 35 cm/sec    (2) hGH Content

Microcapsule (10 mg) was weighed precisely in a 5 ml messflask,acetonitrile (1.75 ml) was added thereto and the mixture wasultra-sonicated. To the obtained acetonitrile solution was added 150 mMphosphate saline buffer (pH 8.0, 3 ml), and the solution wasultra-sonicated and made up to a given volume with 150 mM phosphatesaline buffer (pH 8.0). An 1 ml portion of the solution was centrifugedat 15000 rpm for 10 min, and the supernatant was filtered using amembrane filter having the pore size of 0.5 μm. This hGH extractsolution was then subjected to size exclusion high-performance liquidchromatography under the following conditions to measure the content ofhGH.

-   Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured by Tosoh    Corporation)-   Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution-   Flow rate: 0.6 ml/min    The results are shown in Table 1.

TABLE 1 Contents of residual dichloromethane and hGH in microcapsulesConditions for treatment Quality Tempera Residual Content Pressure tureTime DCM of hGH (Mpa) (° C.) (min) (ppm) (%) Untreated microcapsules 73810.61 (1) 2 15 15 137 10.50 (2) 2 15 30  50 10.46 (3) 2 15 45 <32 10.41(4) 2 15 60 <32 10.52

As seen from the results in Table 1, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in ahigh-pressure gaseous state markedly decreased in comparison with theuntreated microcapsules. Furthermore, it was found that the content ofhGH in the microcapsules did not decrease by the treatment with carbondioxide in a high-pressure gaseous state.

TEST EXAMPLE 2

For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 1 (1) to (4), the amount of hGHaggregate and hGH related protein in the microcapsules were measured bythe following method.

(1) hGH Aggregate

Microcapsules (10 mg) were weighted precisely, acetonitrile (2.5 ml) wasadded thereto, and the sample was dispersed by ultrasonic irradiation.The sample was subsequently irradiated with ultrasonic for about 2 min,and centrifuged at 3000 rpm for 10 minutes. The supernatant was removed,and acetonitrile (2.5 ml) was added to the residue. The residue wasdispersed by ultrasonic irradiation. The dispersion was subsequentlyirradiated with ultrasonic for about 2 minutes, and centrifuged at 3000rpm for 10 minutes. The supernatant was removed, and the residue wasdried in a desiccator under reduced pressure. To the residue was added adiluent (phosphate buffer (pH 8.0)/acetonitrile mixed solution (13:7),1.25 ml), and the sample was dispersed by ultrasonic irradiation. Thedispersion was subsequently irradiated with ultrasonic for about 2 minand filtered with a membrane filter having a pore size of 0.5 μm, andthe filtrate was used as a sample solution. Separately, hGH referencestandard (0.2 ml) was added to a diluent (0.2 ml). This solution (0.2ml) was added to a diluent (4.8 ml) to obtain a standardized solution.The sample solution and standardized solution (each 50 μl) were measuredby liquid chromatography under the following conditions, respectively.The peak area of the peak that elutes earlier than the retention time ofhGH in the sample solution and the peak area of hGH in the standardizedsolution were measured by automatic integration to calculate the contentof aggregate. At the same time, a diluent (50 μl) was injected and thepeak detected in blank was subtracted from the calculation.

-   Detector: ultraviolet spectrometer (wavelength for measurement: 214    nm)-   Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured by Tosoh    Corporation)-   Column temperature: constant temperature about 25° C.-   Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution-   Flow rate: 0.6 ml/min    (2) hGH Related Protein

Microcapsules (40 mg) were precisely weighed and acetonitrile (2 ml) wasadded thereto. The sample was dispersed by ultrasonic irradiation. Thedispersion was subsequently irradiated with ultrasonic for about 2minutes. Phosphate buffer (pH8.0, 3 ml) was added thereto and thedispersion was irradiated with ultrasonic for about 2 minutes withoccasionally shaking, and centrifuged at 4° C. for 3500 rpm for 10minutes. The supernatant was filtered with a membrane filter having poresize of 0.5 μm, and the filtrate was used as a sample solution.Separately, hGH reference standard (0.1 ml) was added to a diluent(phosphate buffer (pH 8.0)/acetonitrile mixed solution (13:7), 3.9 ml)to obtain a standardized solution. The sample solution and standardizedsolution (each 20 μl) were measured by liquid chromatography under thefollowing conditions, respectively. The peak areas of the substancesother than hGH in the sample solution and the peak area of hGH in thestandardized solution were measured by automatic integration,respectively, to calculate the content of related protein. At the sametime, a diluent (20 μl) was injected and the peak detected in blank wassubtracted from the calculation.

-   Detector: ultraviolet spectrometer (measurement wavelength: 220 nm)-   Column: PROTEIN C4, 4.6 mm i.d.×250 mm (VYDAC)-   Column temperature: constant temperature about 45° C.-   Mobile phase:(A) 2-amino-2-hydroxymethyl-1,3-propanediol buffer (pH    7.5)/1-propanol mixed solution (19:6)-   (B) 2-amino-2-hydroxymethyl-1,3-propanediol buffer (pH    7.5)/1-propanol mixed solution (17:8)

The solution (A) and solution (B) were flowed in the proportion of(1:1).

-   Flow rate: 0.5 ml/min

The results are shown in Table 2.

TABLE 2 Contents of hGH aggregate and related protein in microcapsulesConditions for treatment Quality Tempera Aggre- Related Pressure tureTime gate protein (Mpa) (° C.) (min) (%) (%) Untreated microcapsules1.95 5.08 (1) 2 15 15 2.00 5.42 (2) 2 15 30 1.96 5.47 (3) 2 15 45 1.945.50 (4) 2 15 60 1.95 5.26

As seen from the results in Table 2, the amounts of hGH aggregate andrelated protein in the microcapsules treated with carbon doixide in thestate of high-pressure gas did not increase in comparison with those ofthe untreated microcapsules.

TEST EXAMPLE 3

For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 1 (1) to (4), the mean particlediameter and in vivo initial release percentage of the microcapsuleswere measured by the following method.

(1) Mean Particle Diameter of Microcapsules

The mean particle diameter of microcapsules was measured using ameasurement apparatus for particle size distribution (Multisizer II,Coulter Electronics Ltd., Beds, UK).

(2) In vivo Initial Release Percentage

Rats were subjected to immunosuppression treatment with tacrolimus.Prograf injection (manufactured by Fujisawa Pharmaceutical Co., Ltd., 5mg) was diluted with saline. The dilution was subcutaneouslyadministered at the dose of, 0.4 mg/0.2 ml/rat (three days before thefirst administration of the microcapsules), 0.2 mg/0.2 ml/rat(immediately after the first administration of microcapsules, and on the4th, 7th and 11th days after administration), 0.3 mg/0.2 ml/rat (on the14th, 18th, 21st, 25th, 28th and 32nd days after the firstadministration), respectively, whereby the production of antibodies tohGH could be suppressed, which allowed the evaluation of theconcentration of hGH in the blood serum of rats for 5 weeks after thefirst administration.

The microcapsules were dispersed in a dispersion medium (5% mannitol,0.5% carboxymethylcellulose, 0.1% Tween80) at the concentration of 16 mghGH/ml. The obtained dispersion (0.75 ml) was subcutaneous administeredto the back of the rat under ether anesthesia. The dose was 12 mg ashGH. After the administration of the microcapsules, blood was collectedwith time from the caudal vein and blood serum was fractionated.

The measurement of the concentration of hGH in blood serum was measuredby immunoradiometric assay (Ab beads HGH, manufactured by Eiken ChemicalCo., Ltd.).

To the immunosuppressed rats were subcutaneously administered a solutionof hGH at the dose of 5, 10 and 20 mg/kg, respectively, and blood wascollected with time and the concentration of hGH in blood serum wasmeasured. AUC was calculated by trapezoid method. From the AUC up to 24hours after administration of microcapsules, the administered amount ofhGH, the corresponding administered amount of hGH solution in the caseof subcutaneous administration was calculated, which was divided by theadministered amount of microcapsules (12 mg) to calculate the initialrelease percentage.

The results are shown in Table 3.

TABLE 3 Mean particle diameter and initial release percentage ofmicrocapsules Quality Conditions for treatment Mean Initial Temperaparticle release Pressure ture Time diameter percen- (Mpa) (° C.) (min)(μm) tage (%) Untreated microcapsules 36.2 28.3 (1) 2 15 15 34.9 17.0(2) 2 15 30 35.7 16.7 (3) 2 15 45 35.1 13.8 (4) 2 15 60 37.7 24.2

As seen from the results in Table 3, it was confirmed that the meanparticle diameter of the microcapsules did not change by the treatmentwith carbon dioxide in a high-pressure gaseous state and did notaggregate. Furthermore, the initial release percentage of themicrocapsules treated with carbon dioxide in a high-pressure gaseousstate markedly decreased in comparison with that of the untreatedmicrocapsules.

TEST EXAMPLE 4

For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 2 (1) to (4), the amount of residualdichloromethane (DCM) and hGH content in the microcapsules were measuredby the following method.

(1) Amount of Residual Dichloromethane (DCM)

Microcapsules (about 100 mg) were weighed precisely, dissolved indimethylsulfoxide to made up to exactly 5 ml to prepare a samplesolution. Separately, dichloromethane (about 1 g) was measuredprecisely, and dimethylsulfoxide was added thereto to made up to exactly20 ml. This solution was diluted by exactly 10000 times withdimethylsulfoxide to obtain a standardized solution. The sample solutionand standardized solution (each 1 μl) were tested by gas chromatographyunder the following conditions, and the peak area of dichloromethane foreach solution was measured by automatic integration to calculate theamount of dichloromethane.

-   Detector: hydrogen flame ionization detector-   Column: OVI-G43 film thickness 3 μm, 0.53 mm i.d.×30 m (Supelco)-   Inlet temperature: 140° C.-   Detector temperature: 260° C.-   Column temperature: 40° C. (10 min retention)→240° C. (35°    C./min)→240° C. (20 min retention)→cooling→40° C.-   Carrier gas: helium-   Flow: 35 cm/sec    (2) hGH Content

Microcapsules (20 mg) were weighed precisely in a 5 ml graduated flask,and acetonitrile (1.75 ml) was added thereto and the mixture wasultra-sonicated. To the obtained acetonitrile solution was added 150 mMphosphate saline buffer (pH 8.0, 3 ml) and the solution wasultra-sonicated. To the solution was added 150 mM phosphate salinebuffer (pH 8.0) to make up to a given volume. A 1 ml portion of thesolution was centrifuged at 15000 rpm for 10 min, and the supernatantwas filtered using a membrane filter having the pore size of 0.5 μm.This hGH extract solution was then subjected to size exclusionhigh-performance liquid chromatography under the following conditions tomeasure the content of hGH.

-   Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured by Tosoh    Corporation)-   Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution-   Flow rate: 0.6 ml/min    The results are shown in Table 4.

TABLE 4 Contents of residual dichioromethane and hGH in microcapsulesConditions for treatment Quality Tempera Residual Content Pressure tureTime DCM of hGH (Mpa) (° C.) (min) (ppm) (%) Untreated microcapsules4283 4.82 (1) 2 15 30 683 4.72 (2) 2 15 60 207 4.74 (3) 2 15 180 26 4.68(4) 1 15 720 1000 4.74

As is seen from the results in Table 4, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in ahigh-pressure gaseous state markedly decreased in comparison with thatof the untreated microcapsules. Furthermore, it was found that thecontent of hGH in the microcapsules was not decreased by the treatmentwith carbon dioxide in a high-pressure gaseous state.

TEST EXAMPLE 5

For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 2 (1) to (4), the amount of hGHaggregate and hGH related protein in the microcapsules were measured bythe following method.

(1) hGH Aggregate

Microcapsules (10 mg) were weighed precisely, acetonitrile (2.5 ml) wasadded thereto, and the sample was dispersed by ultrasonic irradiation.The sample was subsequently irradiated with ultrasonic for about 2minutes, and centrifuged at 3000 rpm for 10 minutes. The supernatant wasremoved, and acetonitrile (2.5 ml) was added to the residue. The residuewas dispersed by ultrasonic irradiation. The dispersion was subsequentlyirradiated with ultrasonic for about 2 min, and centrifuged at 3000 rpmfor 10 minutes. The supernatant was removed, and the residue was driedin a desiccator under reduced pressure. To the residue was added adiluent (phosphate buffer (pH 8.0)/acetonitrile mixed solution (13:7),1.25 ml), and the sample was dispersed by ultrasonic irradiation. Thedispersion was subsequently irradiated with ultrasonic for about 2minutes and filtered with a membrane filter having pore size of 0.5 μm,and the filtrate was used as a sample solution. Separately, hGHreference standard (0.2 ml) was added to a diluent (0.2 ml). Thissolution (0.2 ml) was added to a diluent (4.8 ml) to obtain astandardized solution. The sample solution and standardized solution(each 50 μl) were measured by liquid chromatography under the followingconditions, respectively. The peak area of the peak that eluted earlierthan the retention time of hGH in the sample solution and the peak areaof hGH in the standardized solution were measured by automaticintegration to calculate the content of aggregate. At the same time, adiluent (50 μl) was injected and the peak detected in blank wassubtracted from the calculation.

-   Detector: ultraviolet spectrometer (wavelength for measurement: 214    nm)-   Column: TSK-gel G2000SWXL, 7.8 mm i.d.×300 mm (manufactured by Tosoh    Corporation)-   Column temperature: constant temperature about 25° C.-   Mobile phase: 0.05 mol/l ammonium hydrogencarbonate solution-   Flow rate: 0.6 ml/min    (2) hGH Related Protein

Microcapsules (40 mg) were precisely weighed and acetonitrile (2 ml) wasadded thereto. The sample was dispersed by ultrasonic irradiation. Thedispersion was subsequently irradiated with ultrasonic for about 2minutes. Phosphate buffer (pH 8.0, 3 ml) was added thereto and thedispersion was irradiated with ultrasonic for about 2 minutes withoccasionally shaking, and centrifuged at 4° C. for 3500 rpm for 10 min.The supernatant was filtered with a membrane filter having pore size of0.5 μm, and the filtrate was used as a sample solution. Separately, hGHreference standard (0.1 ml) was added to a diluent (phosphate buffer (pH8.0)/acetonitrile mixed solution (13:7), 3.9 ml) to obtain astandardized solution. The sample solution and standardized solution(each 20 μl) were measured by liquid chromatography under the followingconditions, respectively. The peak areas of the substances other thanhGH in the sample solution and the peak area of hGH in the standardizedsolution were measured by automatic integration, respectively, tocalculate the content of related protein. At the same time, a diluent(20 μl) was injected and the peak detected in blank was subtracted fromthe calculation.

-   Detector: ultraviolet spectrometer (measurement wavelength: 220 nm)-   Column: PROTEIN C4, 4.6 mm i.d.×250 mm (VYDAC)-   Column temperature: constant temperature about 45° C.-   Mobile phase: (A) 2-amino-2-hydroxymethyl-1,3-propanediol buffer (ph    7.5)/1-propanol mixed solution (19:6)-   (B) 2-amino-2-hydroxymethyl-1,3-propanediol buffer (ph    7.5)/1-propanol mixed solution (17:8)

The solution (A) and solution (B) were flowed in the proportion of(1:1).

-   Flow rate: 0.5 ml/min

The results are shown in Table 5.

TABLE 5 Contents of hGH aggregate and related protein in microcapsulesConditions for treatment Quality Temper- Aggre- Relalted Pressure atureTime gate protein (Mpa) (° C.) (min) (ppm) (%) Untreated microcapsules1.44 7.46 (1) 2 15 30 1.29 7.90 (2) 2 15 60 1.24 7.44 (3) 2 15 180 1.387.48 (4) 1 15 720 1.48 7.54

As is seen from the results in Table 5, the amounts of hGH aggregate andrelated protein in the microcapsules treated with carbon dioxide in thehigh-pressure gaseous state did not increase in comparison with those ofthe untreated microcapsules.

TEST EXAMPLE 6

For the hGH-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 2 (1) to (4), the mean particlediameter and in vivo initial release percentage of the microcapsuleswere measured by the following method.

(1) Mean Particle Diameter of Microcapsules

The mean particle diameter of microcapsules was measured using ameasurement apparatus for particle size distribution (Multisizer II,Coulter Electronics Ltd., Beds, UK).

(2) In vivo Initial Release Percentage

Rats were subjected to immunosuppression treatment with tacrolimus.Prograf injection (manufactured by Fujisawa Pharmaceutical Co., Ltd., 5mg) was diluted with saline. The dilution was subcutaneouslyadministered in the dose of, 0.4 mg/0.2 ml/rat (three days beforeadministration of the microcapsules), 0.2 mg/0.2 ml/rat (immediatelyafter the first administration of microcapsules, and on the 4th, 7th,11th, 14th and 18th days after the first administration), respectively.The microcapsules were dispersed in a dispersion medium (5% mannitol,0.5% carboxymethylcellulose, 0.1% Tween80) at the concentration of 8 mghGH/ml. The obtained dispersion (0.75 ml) was subcutaneous administeredto the back of the rat under ether anesthesia. The dose was 6 mg as hGH.After the administration of the microcapsules, blood was sequentiallytaken from the caudal vein and blood serum was fractionated.

The concentration of hGH in blood serum was measured byimmunoradiometric assay (Ab beads HGH, Eiken Chemical Co., Ltd.).

To the immunosuppressed rat was subcutaneously administered a solutionof hGH at the dose of 5, 10 and 20 mg/kg, respectively, and blood wascollected with time and the concentration of hGH in blood serum wasmeasured. AUC was calculated by trapezoid method. From the AUC up to 24hr after administration of microcapsules, the administered amount ofhGH, the corresponding administered amount of hGH solution in the caseof subcutaneous administration was calculated, which was divided by theadministered amount of microcapsules (6 mg) to calculate the initialrelease percentage.

The results are shown in Table 6.

TABLE 6 Mean particle diameter and initial release percentage ofmicrocapsules Quality Conditions for treatment Mean Initial Temper-particle release Pressure ature Time diameter percent- (Mpa) (° C.)(min) (μm) age (%) Untreated microcapsules 36.6 25.8 (1) 2 15 30 37.913.9 (2) 2 15 60 37.2 15.0 (3) 2 15 180 37.9 19.0 (4) 1 15 720 37.3 14.7

As is seen from the results in Table 6, it was confirmed that the meanparticle diameter of the microcapsules was not changed by the treatmentwith carbon dioxide in the high-pressure gaseous state and themicrocapsules did not aggregate. Furthermore, the initial releasepercentage of the microcapsules treated with carbon dioxide in thehigh-pressure gaseous state markedly decreased in comparison with thatof the untreated microcapsules.

TEST EXAMPLE 7

For the candesartan-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 3 (1) to (18), the amount of residualdichloromethane (DCM) and candesartan content in the microcapsules weremeasured by the following method.

(1) Amount of Residual Dichloromethane (DCM)

Microcapsules (about 100 mg) were weighed precisely, dissolved indimethylsulfoxide to make up to exactly 5 ml to prepare a samplesolution. Separately, dichloromethane (about 1 g) was weighed precisely,and dimethylsulfoxide was added thereto to make up to exactly 20 ml.This solution was diluted by exactly 10000 times with dimethylsulfoxideto obtain a standardized solution. The sample solution and standardizedsolution (each 1 μl) were tested by gas chromatography under thefollowing conditions, and the peak area of dichloromethane for eachsolution was measured by automatic integration to calculate the amountof dichloromethane.

-   Detector: hydrogen flame ionization detector-   Column: OVI-G43 film thickness 3 μm, 0.53 mm i.d.×30 m (Supelco)-   Inlet temperature: 140° C.-   Detector temperature: 260° C.-   Column temperature: 40° C. (10 min retention)→260° C. (35° C./min)    (10 min retention)-   Carrier gas: helium-   Flow: 35 cm/sec    (2) Candesartan Content

Microcapsules (5 to 10 mg) were weighed precisely in a centrifuge tube,HPLC mobile phase (30 ml) was added thereto and the mixture was stirredwith shaking for 1 hour. The mixture was then centrifuged at 2950 rpmfor 10 minutes, and the supernatant was filtered with a membrane filterhaving the pore size of 0.5 μm. This candesartan extract solution wasthen subjected to reverse phase high-performance liquid chromatographyunder the following conditions to measure the content of candesartan.

-   Column: Inertsil ODS-3 (4.6 mm×150 mm, manufactured by GL science)-   Mobile phase: 0.1M KH₂PO₄/AcCN/MeOH/AcOH=50/35/15/1 (v/v)-   Flow rate:1 ml/min-   Detection: UV wavelength 254 nm    The results are shown in Table 7.

TABLE 7 Contents of residual dichloromethane and candesartan inmicrocapsules Quality Conditions for treatment Residual Tempera- Chargeddichloro- Drug Pressure ture Time amount methane content (Mpa) (° C.)(min) (g) (ppm) (%) Untreated microcapsules 18026 26.5 (1) 2.0 15 30 0.3826 26.0 (2) 2.0 15 60 0.3 230 26.0 (3) 2.0 15 120 0.3 106 26.5 (4) 2.015 180 0.3 0 26.3 (5) 2.5 15 30 0.3 411 26.1 (6) 2.5 15 60 0.3 375 26.9(7) 2.5 15 120 0.3 0 26.9 (8) 2.5 15 180 0.3 0 26.5 (9) 3.0 15 15 0.36923 26.8 (10) 3.0 15 30 0.3 2993 26.2 (11) 3.0 15 60 0.3 0 26.8 (12)3.0 15 120 0.3 0 26.9 (13) 3.0 15 180 0.3 0 26.8 (14) 3.5 15 30 0.3 392626.3 (15) 3.5 15 60 0.3 0 27.1 (16) 3.5 15 120 0.3 321 26.9 (17) 3.5 15180 0.3 0 26.5 (18) 4.0 15 30 0.3 1148 27.1

As is seen from the results in Table 7, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in thehigh-pressure gaseous state markedly decreased in comparison with thatof the untreated microcapsules. Furthermore, it was confirmed that thecontent of candesartan in the microcapsules was not decreased by thetreatment with carbon dioxide in the state of high-pressure gas.

TEST EXAMPLE 8

For the candesartan-containing microcapsules and untreated lyophilizedmicrocapsules obtained in Example 4 (1) to (3), the amount of residualdichloromethane (DCM) and candesartan content in the microcapsules weremeasured by the similar method to that of Test Example 7.

The results are shown in Table 8.

TABLE 8 Contents of residual dichloromethane and candesartan inmicrocapsules Quality Conditions for treatment Residual Tempera- Chargeddichloro- Drug Pressure ture Time amount methane content (Mpa) (° C.)(min) (g) (ppm) (%) Untreated microcapsules 24692 26.4 (1) 3.0 15 60 0.3116 26.8 (2) 3.0 15 60 2 0 26.4 (3) 3.0 15 60 5 137 27.7

As is seen from the results in Table 8, the amount of residualdichloromethane in the microcapsules treated with carbon dioxide in thehigh-pressure gaseous state markedly decreased in comparison with thatof the untreated microcapsules. Furthermore, it was confirmed that thecontent of candesartan in the microcapsules did not decrease by thetreatment with carbon dioxide in a high-pressure gaseous state.

INDUSTRIAL APPLICABILITY

According to the present invention, in a method for producing asustained-release preparation, by forming a solid material containing abioactive substance and a polymer and contacting the solid material withhigh-pressure gas, it is possible to produce a sustained-releasepreparation which is a medicament having such very superior clinicalproperties that the excess amount of initial release of the bioactivesubstance immediately after administration is markedly suppressed, aconstant amount of the bioactive substance is being released fromimmediately after administration over a long period of time, and thedenaturation of the bioactive substance and the residual organic solventare extremely decreased. Furthermore, by modifying the method forremoving a solvent, the treatment period required for the removal of thesolvent has been markedly decreased.

1. A method for producing sustained-release microcapsules containing anon-peptidic bioactive substance, which comprises forming a solidmaterial containing the non-peptidic bioactive substance and a polymerby (a) in-water drying method, (b) phase separation method or (c)spray-drying method, and then contacting the solid material with carbondioxide to form the sustained-release microcapsules, wherein thepressure of the carbon dioxide is 1 MPa to 7 MPa at a temperature rangeof −60° C. to +20° C. based on the glass transition temperature of thepolymer, with the proviso that the solid material is not in contact withthe carbon dioxide before or during formation of the solid material. 2.The method according to claim 1, wherein the non-peptidic bioactivesubstance is unstable to heat or solvents.
 3. The method according toclaim 1, wherein the non-peptidic compound is a compound having anoxygen atom in the molecule.
 4. The method according to claim 1, whereinthe non-peptidic compound is a compound having an ether bond or acarbonyl group.
 5. The method according to claim 1, wherein thenon-peptidic compound is a compound represented by the formula (I):

wherein R¹ represents a group capable of forming an anion or a groupwhich may be converted into said group, X represents that the phenylenegroup and the phenyl group are linked directly or via a spacer of anatomic chain having two or less atom(s), n represents an integer of 1 or2, ring A represents a benzene ring which may be further substituted, R²represents a group capable of forming an anion or a group which may beconverted into said group, R³ represents a hydrocarbon residue which maylink via a heteroatom and may be substituted, or a salt thereof.
 6. Themethod according to claim 1, wherein the non-peptidic compound islosartan, eprosartan, candesartan cilexetil, candesartan, valsartan,telmisartan, irbesartan, tasosartan or olmesartan.
 7. The methodaccording to claim 1, wherein the non-peptidic compound is candesartan.8. The method according to claim 1, wherein the polymer isbiodegradable.
 9. The method according to claim 8, wherein thebiodegradable polymer is a homopolymer or a copolymer ofα-hydroxycarboxylic acids, or a mixture thereof.
 10. The methodaccording to claim 9, wherein the biodegradable polymer is a homopolymeror a copolymer of lactic acid/glycolic acid having a composition ratioof lactic acid/glycolic acid of 100/0 to 40/60 mol %.
 11. The methodaccording to claim 9, wherein the biodegradable polymer is a homopolymerof lactic acid.
 12. The method according to claim 8, wherein theweight-average molecular weight of the biodegradable polymer is 3,000 to50,000.
 13. The method according to claim 1, wherein the solid materialis contacted with carbon dioxide, wherein the pressure of the carbondioxide is 1 MPa to 7 MPa at a temperature range of −40° C. to +0° C.based on the glass transition temperature of the polymer.
 14. The methodaccording to claim 1, wherein the period for contacting the solidmaterial with carbon dioxide is 5 minutes to 48 hours.
 15. The methodaccording to claim 14, wherein the period for contacting the solidmaterial with carbon dioxide is 10 minutes to 12 hours.
 16. The methodaccording to claim 1, wherein the carbon dioxide is inert to thenon-peptidic bioactive substance and polymer.
 17. The method accordingto claim 1, wherein the pressure of the carbon dioxide is 1 MPa to 4MPa.
 18. The method according to claim 1, wherein the solid material isobtained by in-water drying method.